Combined Use of a Solid-Phase Hexapeptide Ligand Library with Liquid Chromatography and Two-Dimensional Difference Gel Electrophoresis for Intact Plasma Proteomics
Hindawi Publishing CorporationCopyright Hindawi Publishing Corporation

Tatsuo Hagiwara
Division of Pharmacoproteomics
National Cancer Center Research Institute
Chuo-ku104-0045TokyoJapan

Department of Biological Science and Technology
Laboratory of Genome Biology
Tokyo University of Science
278-8510TokyoJapan

Yumi Saito
Division of Pharmacoproteomics
National Cancer Center Research Institute
Chuo-ku104-0045TokyoJapan

Yukiko Nakamura
Division of Pharmacoproteomics
National Cancer Center Research Institute
Chuo-ku104-0045TokyoJapan

Laboratory of Proteome Research
National Institute of Biomedical Innovation
567-0085OsakaJapan

Takeshi Tomonaga
Laboratory of Proteome Research
National Institute of Biomedical Innovation
567-0085OsakaJapan

Yasufumi Murakami
Department of Biological Science and Technology
Laboratory of Genome Biology
Tokyo University of Science
278-8510TokyoJapan

Tadashi Kondo
Division of Pharmacoproteomics
National Cancer Center Research Institute
Chuo-ku104-0045TokyoJapan

David E Misek
Combined Use of a Solid-Phase Hexapeptide Ligand Library with Liquid Chromatography and Two-Dimensional Difference Gel Electrophoresis for Intact Plasma Proteomics

International Journal of Proteomics
Hindawi Publishing Corporation201111Received 2 May 2011; Accepted 9 June 2011Research Article Correspondence should be addressed to Tadashi Kondo, email@example.com Academic Editor:
The intact plasma proteome is of great interest in biomarker studies because intact proteins reflect posttranslational protein processing such as phosphorylation that may correspond to disease status. We examined the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins. Plasma proteins were sequentially fractionated using depletion columns for albumin and immunoglobulin, and separated using an anion-exchange column. Proteins in each fraction were treated with a solid-phase hexapeptide ligand library and compared to those without treatment. Two-dimensional difference gel electrophoresis demonstrated an increased number of protein spots in the treated samples. Mass spectrometric studies of these protein spots with unique intensity in the treated samples resulted in the identification of high-and medium-abundance proteins. Our results demonstrated the possible utility of a solidphase hexapeptide ligand library to reveal greater number of intact plasma proteins. The characteristics of proteins with unique affinity to the library remain to be clarified by more extensive mass spectrometric protein identification, and optimized protocols should be established for large-scale plasma biomarker studies.

Introduction

The plasma proteome has been extensively investigated with the aim of biomarker development [1,2]. Plasma is the most accessible clinical material, and plasma biomarkers for early diagnosis and monitoring the response to therapy and disease recurrence would be beneficial for patients with cancer. Because proteins released by tumors, particularly early-stage tumors, are expected to exist in very low concentrations and plasma contains various proteins with considerable heterogeneity between and within patients, the identification of novel plasma biomarkers represents a substantial challenge.

Global expression studies on intact plasma proteins are of special interest in biomarker studies as the intact proteins reflect the functional features of protein structure. Those include posttranslational processing such as phosphorylation and glycosylation. Peptide subsets from complex digests have been analyzed for plasma proteomics, resulting in the identification of low-abundance proteins such as tissue leakage proteins [3] and biomarker candidates [4]. However, analysis of peptide digests may not be sensitive to posttranslational protein processing, and may therefore not reveal many relevant protein isoforms associated with disease status. To date, much effort has been devoted to detect trace intact proteins in complex plasma samples.

The utility of a combinatorial hexapeptide ligand library immobilized on a solid-phase matrix has been reported, introduced to intact plasma proteomics [5][6][7][8][9], and 2 International Journal of Proteomics commercialized as ProteoMiner (Bio-Rad Laboratories, Hercules, CA, USA). ProteoMiner contains millions of randomly synthesized hexapeptide ligands that are equally represented with a selected number of targets. When a complex plasma protein extract is exposed, the hexapeptide ligands for highabundance proteins are saturated, but the majority remains unbound. In contrast, the proteins which do not saturate the corresponding hexapeptide ligands and usually not observed by the conventional methods will appear in the proteome data. The approach of using a combinatorial hexapeptide ligand library is different from that of using depletion and separation; thus, it reveals a novel aspect of the plasma proteome. A recent report demonstrated that prefractionation using a hexapeptide ligand library for shotgun mass spectrometry studies identified plasma proteins not recorded in the Human Plasma Proteome Project [10]. The combined use of a hexapeptide ligand library with depletion and separation methods has also been a challenge in deeper plasma proteomics [11], and the resulting protein contents are examined by gel electrophoresis and mass spectrometry [12,13]. ProteoMiner has been used for disease biomarker studies in lung cancer [14] and liver cancer [15]. Considering that it will potentially visualize the unique plasma proteome aspects, the application and optimization of a solid-phase hexapeptide ligand library for disease biomarker studies should be further investigated.

In this study, we examined the utility of a solid-phase hexapeptide ligand library in combination with a depletion column, an anion-exchange column, and 2D-DIGE that allows an instant visual comparison of the protein patterns. Protein spots exhibiting prominent differences between samples treated with and without the library were subjected to mass spectrometry. Our study clearly demonstrated that the combined use of the ProteoMiner and the other proteomics modalities can visualize unique plasma proteome.

Materials and Methods

Sample Preparation.

Frozen human plasma was purchased from Cosmobio KOJ (Tokyo, Japan). After the plasma was placed on ice, 40 mL plasma was centrifuged and 30 mL supernatant was recovered for the following experiments.

Albumin Depletion.

Albumin and other proteins were separated using a HiTrap Blue HP column (5 mL resin, GE, Uppsala, Sweden) with the AKTA Explorer system (GE) at a flow rate of 1.0 mL/min. The separation was initiated by washing the column with rinse buffer (50 mM KH 2 PO 4 /Na 2 HPO 4 , pH 7.0) for 5 min. Plasma (30 mL) was diluted with 60 mL 50 mM KH 2 PO 4 /Na 2 HPO 4 (pH 7.0), and 9 mL of the diluted plasma was injected. The column was then washed with binding buffer (50 mM KH 2 PO 4 /Na 2 HPO 4 , pH 7.0) for 35 min, and the flowthrough fraction was collected. Bound proteins were eluted from the column with elution buffer (50 mM KHPO 4 , 1.5 M KCl, pH 7.0) for 45 min, and the bound fraction was collected. The column was neutralized with rinse buffer for 20 min. This process was repeated 10 times for a total of 90 mL of diluted plasma.

One-third of the flow-through and bound fractions, approximately 150 mL of each, was concentrated to 1.2 mL using a VIVA Spin 20 column (10 K MWCO, 20 mL capacity, Sartorius, Gotteingen, Germany). Then, 1.0 mL and 0.20 mL of the concentrated samples were subjected to treatment with the solid-phase hexapeptide ligand library and 2D-DIGE, respectively. Two-thirds of the flow-through fraction, approximately 300 mL, was subjected to an immunodepletion column.

Immunoglobulin Depletion.

Immunoglobulin was depleted using the HiTrap Protein G HP column (1 mL resin, GE) with the AKTA Explorer system (GE) at a flow rate of 1.0 mL/min. The depletion was initiated by washing the column with rinse buffer (50 mM KH 2 PO 4 /Na 2 HPO 4 , pH 7.0) for 4 min. After 15 mL of the flow-through fraction from the HiTrap Blue HP column was injected, the column was washed with binding buffer (50 mM KH 2 PO 4 /Na 2 HPO 4 , pH 7.0) for 5 min, and the flow-through fraction was collected. Bound proteins were eluted from the column with elution buffer (0.1 M glycine-HCl, pH 2.2) for 8 min and collected as the bound fraction. The collected bound fraction was immediately neutralized with neutralizing buffer (1.0 M Tris-HCl, pH 9.0). The column was equilibrated with rinse buffer for 5 min for reuse. This process was repeated 20 times for a total of two-thirds of the flow-through fraction from the HiTrap Blue HP column (approximately 300 mL).

Half of the flow-through and bound fractions (approximately 200 mL and 80 mL, resp.) were concentrated to 1.2 mL and 0.25 mL, respectively, using VIVA Spin 20 columns (Sartorius). Then, 1.0 mL of the concentrated flowthrough fraction and 0.20 mL of the concentrated bound fraction were subjected to treatment with the solid-phase ligand library, and the remaining samples were subjected to 2D-DIGE. Another half of the flow-through fraction (approximately 200 mL) was concentrated to 2.0 mL using the VIVA Spin 20 column (Sartorius). After diluting with 38 mL of 25 mM Tris-HCl (pH 9.0), the sample was subjected to separation on an anion-exchange column.

Anion

Exchange. The flow-through fraction from the HiTrap Protein G HP column was separated using the Resource Q column (1 mL resin, 6.4 mm id × 30 mm, GE) with the AKTA Explorer system (GE) at a flow rate of 3.0 mL/min. The separation was initiated by washing the column with rinse buffer (25 mM Tris-HCl, pH 9.0) for 4 min, and 5 mL of the flow-through fraction from the HiTrap Protein G HP column was injected. The separations were performed using a stepwise NaCl gradient as follows: 0, 100, 150, 200, 250, and 1000 mM for 5 min each. All samples contained 25 mM Tris-HCl, pH 9.0. The column was washed with rinse buffer (25 mM Tris-HCl, pH 9.0) for 5 min. This process was repeated 8 times for a total of 40 mL of the diluted flow-through fraction from the HiTrap Protein G HP column.

The collected samples were concentrated to 0.25 mL, and the buffer was exchanged gradually with 25 mM Tris-HCl (pH 9.0) using the VIVA Spin 20 column (Sartorius). Then, International Journal of Proteomics 3 0.2 mL and 0.05 mL were subjected to treatment with the solid-phase ligand library and 2D-DIGE, respectively.

Treatment with the Solid-Phase Ligand Library.

A solidphase combinatorial library of hexapeptides was purchased from Bio-Rad Laboratories (ProteoMiner kit). Unprocessed plasma (1 mL) and the flow-through fractions from the HiTrap Blue HP and HiTrap Protein G HP columns were treated using the ProteoMiner large-capacity kit, and 0.2 mL of the bound fraction from the HiTrap Protein G HP column, and all fractions from the Resource Q column were treated using the ProteoMiner small-capacity kit. After 2 h of incubation at room temperature, the unbound fraction was washed out by centrifugation. After rinsing, the bound sample was eluted with an elution reagent containing 8 M urea, 2% CHAPS, and 5% acetic acid, according to the manufacturer's instructions.

Measurement of Protein Concentration.

Protein concentration was measured using a protein assay kit (Bio-Rad), according to the manufacturer's instructions (Table 1).

SDS-PAGE.

Protein samples (1 μg) were examined by electrophoresis using 18-well precast 12.5% polyacrylamide gel plates (e-PAGEL, ATTO, Tokyo, Japan). Electrophoresis was performed at a constant current of 40 mA for 80 min and using the page Run AE6531 system [16]. Silver staining was performed using the Silver Stain KANTO III kit (Kanto Chemical, Tokyo, Japan), according to the manufacturer's instructions.

2D-DIGE.

2D-DIGE was performed as described previously [17]. Briefly, protein samples (20 μg) were labeled with the Cy3 or Cy5 fluorescent dye (CyDye DIGE Fluor saturation dye, GE), and differentially labeled protein samples were mixed. After dividing into 3, the labeled protein samples were separated by 2D-PAGE. The first-dimension separation was performed using a 24 cm length immobiline gel (IPG, pI 4-7, GE) and Multiphor II (GE) whereas the second-dimension separation was performed using gradient gels prepared in house and EttanDalttwelve (GE). The gels were scanned using a laser scanner (Typhoon Trio, GE) at an appropriate wavelength for Cy3 or Cy5. The Cy3 and Cy5 intensities were compared in the same gel using the Progenesis SameSpots software (version 4.0; Nonlinear Dynamics, Newcastle, UK). ProteoMiner-treated and untreated samples were labeled with Cy3 and Cy5, respectively, or with Cy5 and Cy3, respectively. Six gels were run for each sample. The average value of the intensity ratio was calculated among the triplicate gels for all protein spots and then averaged between the 2 samples for further study. Spot intensity data were exported from the Progenesis SameSpots software as Excel files amenable to numerical data analysis.

Mass Spectrometric Protein Identification.

Proteins were extracted from the protein spots by in-gel digestion, as reported previously [17]. Briefly, protein samples (100 μg) were labeled with Cy3 and separated by 2D-PAGE. The

Results and Discussion

We previously reported the utility of combining multidimensional chromatography and 2D-DIGE for intact plasma proteomics. Extensive fractionation by the different separation modes increased the number of protein spots on 2D-DIGE and allowed a quantitative comparison between the plasma samples from healthy donors and those from patients with lung adenocarcinoma [18] and pancreatic cancer [19]. However, mass spectrometric protein identification revealed that protein spots with a significant difference between the sample groups corresponded to high-and mediumabundance proteins such as acute-phase proteins, but no known plasma tumor markers were detected. Thus, we concluded that further investigations are needed to reveal low-abundance proteins for biomarker studies. In this study, we examined whether a novel technology, a solid-phase hexapeptide ligand library could improve the linkage of multidimensional chromatography and 2D-DIGE.

Overall View of Protein Fractionation: Comparison and

Detection. The overall view of sequential protein separation is shown in Figure 1. A sample equivalent to 10 mL plasma was separated using 3 different columns and then treated with the solid-phase hexapeptide ligand library ProteoMiner. The ProteoMiner-treated and untreated samples were compared using 2D-DIGE by labeling them with different fluorescent dyes and separating the labeled proteins on an identical gel. The protein spots with significantly different intensities between the ProteoMiner-treated and untreated samples were subjected to mass spectrometry to identify the proteins.

The number of observable low-abundance proteins was affected by the initial amount of plasma sample and the sensitivity of the final quantification method. We used a relatively large volume of plasma sample (10 mL) as the initial material. The immunodepletion columns allow the use of only a small volume of plasma sample for separation. Furthermore, a significantly larger number of plasma samples should be examined to obtain conclusive results for biomarker development. A larger volume of samples can be manipulated by repeatedly using the same immunodepletion column. Although it is quite feasible, special attention may be required to maintain reproducibility during a long period of use. In this study, we used Blue Sepharose and Protein G-Sepharose columns in a sequential manner to deplete albumin and subsequently immunoglobulin and to minimize repeated use of the same column. Although these columns may have less sensitivity than an immunodepletion column and deplete nontargeted proteins that may bind to albumin and immunoglobulin, a larger volume of plasma sample can be treated in individual procedures. A previous study indicated that Cibacron Blue beads remove a major portion of the albumin but with concomitant loss of potentially important peptides and proteins [20]. Thus, we examined both the column-bound and flow-through fractions (Figure 1). Although the specificity of Cibacron Blue beads was not validated in this study, as the purpose of Cibacron Blue was to reduce the complexity of plasma sample, it should not be problem.

To avoid possible redundant proteins in the neighboring fractions as much as possible when utilizing the anionexchange column, we used stepwise elution and fractionation; once all proteins were eluted, the next elution buffer was applied to the column (Figure 1). Considering the complexity of the samples and resolution of an anion-exchange column, extensive fractionation with a gradient buffer system may result in redundant contents among the fractions. We employed 6 stepwise fractionations by monitoring the fraction contents using SDS-PAGE (data not shown).

High Reproducibility of Protein Fractionation by Chromatography.

The ultraviolet detection (280 nm) trace for each run demonstrated consistent separation of albumin and immunoglobulin from the depletion and anion-exchange columns. This high reproducibility may suggest the possible utilities of this approach for biomarker studies (Supplementary Figure 1). High quantitative and qualitative reproducibility of the solid-phase hexapeptide ligand library Pro-teoMiner has been confirmed in previous reports [21,22].

Demonstration of the Effects of Fractionation and Dynamic

Range Reduction. We examined the effects of sequential plasma protein fractionation using 3 columns and the reduction of dynamic range by ProteoMiner ( Figure 2). The contents of the fractionated samples were apparently different from each other. Notably, the protein sample bound to the Blue Sepharose and Protein G-Sepharose columns included many proteins that should be different from the targeted proteins, according to their molecular weights. Treatment of the fractionated samples with ProteoMiner enhanced the proteins that were not observed, except for those bound to the Protein G-Sepharose column. Treatment of the bound fraction from the Protein G-Sepharose column with ProteoMiner did not result in a greater number of observable proteins. This may have been due to the low complexity and narrow dynamic range of proteins in the bound fraction from the Protein G-Sepharose column. There was one order of magnitude in concentration difference for the observed protein bands in the ProteoMiner-treated sample. These observations may reflect that the affinity of proteins for the peptide may not be equal and even the number of peptides bound on the beads is equal, the amount of proteins bound to the ProteoMiner may be different  Table 1. The recovery rate from ProteoMiner was between 0.54 and 6.33%, suggesting that a unique population of protein species selectively bound to ProteoMiner. This assumption was supported by the SDS-PAGE data, except the bound fraction from the Protein G-Sepharose column which included only 4 major proteins that were bound to ProteoMiner (Figure 2).
− + − + − + − + − + ProG BD ProG FT Blue BD Blue

Higher Separation of Fractionated Protein Samples and an

Evaluation of the Effects of ProteoMiner Treatment. Although SDS-PAGE separated individual proteins with higher resolution than chromatography in this study, using it for a quantitative comparison in a biomarker study may be troublesome because many protein bands obviously overlapped ( Figure 2). Thus, we subjected the fractionated samples to 2D-DIGE in order to separate the proteins with higher resolution. Bandow compared ProteoMiner-treated and untreated plasma samples using conventional 2D-PAGE and demonstrated substantial differences between unprocessed and immunodepleted plasma samples [11]. In 2D-DIGE, 2 protein samples were labeled with different fluorescent dyes, mixed, and separated by 2D-PAGE. Because the 2 samples were separated on an identical 2D-PAGE, gel-to-gel variation was compensated. In addition, the wide dynamic range of the fluorescent dyes enabled a quantitative comparison. 2D-DIGE has been applied to compare the performance of ProteoMiner with an immunodepletion column [12]. We further extended the evaluation of the utility of ProteoMiner by loading a high amount of protein and examining the proteins separated by an anion-exchange column.

The fluorescent 2D-PAGE images of the ProteoMinertreated and untreated samples were overlaid with different colors, so that the unique protein contents were visualized (Figures 3 and 4). The results of experiments in which the fluorescent dyes were swapped are shown in Supplementary  Figure 2. Consistent with the SDS-PAGE results (Figure 2), Figure 3 demonstrates that the approach involving depletion of high-abundance proteins and multidimensional separation was an effective prefractionation method to increase the number of protein spots, and the use of ProteoMiner treatment also contributed to reveal more plasma proteins. Because these fractionation methods are based on different binding properties of proteins, their combined use revealed additional plasma proteins.

The number of observed protein spots on 2D gel electrophoresis is summarized in Supplementary Table 2. Overall, the total number of protein spots increased by treating the samples with ProteoMiner, except for the bound fraction from the Protein G-Sepharose column. This observation suggests that ProteoMiner may be a useful tool to observe a greater number of protein spots in prefractionated samples.

We compared the protein spots of the samples with and without ProteoMiner treatment (Supplementary Table 3). Depending on the criteria, different numbers of protein spots showed significantly different intensities. Although the total number of protein spots increased by treating the samples with ProteoMiner (Supplementary Table 2), many protein spots revealed decreased intensity with treatment, suggesting the selective enrichment by ProteoMiner.

Mass Spectrometric Identification of Proteins with Different Affinities to ProteoMiner.

To reveal the characteristics of proteins with a particularly high or low affinity to ProteoMiner, among the protein spots with greater than 5-fold differences (Supplementary Table 3), we selected those with the top 10% different intensities between the ProteoMiner-treated and untreated samples in each fraction and subjected them to mass spectrometric identification. A total of 200 protein spots were subjected to mass spectrometry, and a positive identification was obtained for 128 (Supplementary Table 4). A list of the identified proteins is provided in Supplementary Table 5, and data supporting  protein identification are shown in Supplementary Table 6. These 128 protein spots corresponded to 29 unique proteins. Because the fold difference of the protein spots in the bound fraction from the Protein G-Sepharose column was less than 4, we did not examine them. Of the original plasma samples, vitronectin and albumin were most affected by ProteoMiner treatment and disappeared after depletion and fractionation using the anion-exchange column. The other proteins were identified as enriched (or nonenriched) by ProteoMiner treatment. Proteins bound to ProteoMiner have been reported in previous studies in which the proteins were globally identified by mass spectrometry. Dwivedi et al. demonstrated that albumin, alpha 1-antitrypsin, alpha 2-macroglobulin, apolipoprotein A-I, apolipoprotein A-II, haptoglobin-related protein, and serotransferrin have high affinity to ProteoMiner [21]. In addition, Beseme et al. identified apolipoprotein A-IV, apolipoprotein D, apolipoprotein E, ceruloplasmin, complement C3, fibrinogen beta, fibrinogen gamma, ficolin-2, ficolin-3, paroxonase I, prothrombin, transthyretin, and vitronectin [23]. The protein concentrations identified in this study are summarized in Supplementary Table 6. According to the literatures, the identified proteins were classified as high-and mediumabundance proteins. Adiponectin and the carboxypeptidase N catalytic chain are not reported in previous studies, in which ProteoMiner-treated samples were examined by 2D-PAGE and mass spectrometry.

Adiponectin is an adipocytokine [24][25][26][27] and plays a protective role against obesity-related disorders such as metabolic syndrome [28], type 2 diabetes [29], and cardiovascular disease [30]. Low levels of plasma adiponectin are associated with obesity [31] and many types of malignancies such as liver cancer [32], breast cancer [33], pancreatic cancer [34], and endometrial cancer [35]. An epidemiological study suggested that adiponectin is involved in early colorectal carcinogenesis [36], and that a low circulating adiponectin level is correlated with a poor prognosis in patients with colorectal cancer [37]. The molecular backgrounds of these observations may be attributable to the antiproliferative effects of adiponectin on cancer cells [38].

Carboxypeptidase N (CPN), which is also known as kininase I, arginine carboxypeptidase, and anaphylatoxin inactivator, is a zinc finger metalloprotease. It cleaves basic lysine and arginine residues from the carboxy terminal of proteins [39]. CPN is produced in the liver and secreted into the plasma. It modulates the activity of cytokines such as stromal cell-derived factor-1 alpha [40]. The association of CPN1 with malignancy and other diseases has not been reported, and the clinical utility of CPN1 has not been suggested.

The working hypothesis of this study was that the combined use of different separation methods, including a solidphase hexapeptide ligand library, would increase the number of observable proteins, and finally visualize the proteome that may not be observed otherwise. By loading a high amount of protein and using extensive prefractionation techniques prior to using ProteoMiner, trace proteins became visible in SDS-PAGE, and the number of protein spots on International Journal of Proteomics  2D-DIGE increased significantly. This approach may pave a way to a novel strategy for intact plasma proteomics. In contrast, the present results of mass spectrometric protein identification did not support the use of a solid-phase hexapeptide ligand library to enrich low-abundance proteins. It may be because our present approach had 3 limitations. First, mass spectrometric identification was performed for proteins with a greater prominent difference between the samples with or without ProteoMiner treatment, and only 128 of 200 proteins were successfully identified (Supplementary Table 4), probably because of the low protein amount. Proteins with a smaller difference or amount may include trace proteins. Although we optimized the protocols for mass spectrometric protein identification because the sensitivity of the fluorescent dye in the 2D-DIGE was very high, not all protein spots on 2D-DIGE could be identified by mass spectrometry. To evaluate enriched proteins, the complementary use of an LC-MS/MS shotgun approach may be worth considering. Second, proteins from ProteoMiner were recovered by a single-step elution with 8 M urea, 2% CHAPS, and 5% acetic acid, according to the manufacturer's instructions (Bio-Rad). However, because proteins may have interacted with hexapeptide ligand libraries in all possible modes, the absolute elution process may require sequential steps or more stringent buffer conditions such as boiling 10% SDS with 3% DTE [41]. Furthermore, various binding conditions may also be worth considering to capture whole binding proteins [9]. Third, considering the practical use of trace proteins in a biomarker study, we used as much sample as possible for identifying them and examined 10 mL plasma samples as an initial source. However, a larger volume of plasma sample, such as 100 mL, might be needed to collect rare proteins. In practice, such a high volume of plasma is rarely obtained for many cases in biomarker studies, and we may need to optimize the protocols for use of 10 mL plasma. For instance, we identify the biomarker candidates using 100 mL plasma, and using specific antibody against the identified candidate, we will be able to screen a relatively large number of samples with 10 mL volume or less.
7 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k)
The combined use of the ProteoMiner and the proteomic modalities in this study may enable the quantitative comparison for biomarker studies. We demonstrated that the liquid chromatography was quantitatively reproducible (Supplementary Figure 1), and the quantitative reproducibility of the ProteoMiner and 2D-DIGE was previously reported [10,17]. We may further need to examine how the combined use of such reproducible methods generate the results in a reproducible way, considering the degree of differences that we expect between the samples to be compared.

Conclusions

The use of ProteoMiner in combination with conventional proteomic modalities such as depletion and anion-exchange columns significantly enhanced trace proteins on SDS-PAGE and increased the number of protein spots on 2D-DIGE, suggesting that the use of a solid-phase hexapeptide ligand library has great potential for intact plasma proteomics. Mass spectrometric protein identification revealed that high-and middle-abundance proteins were enriched by ProteoMiner, and the characteristics of proteins with unique affinity to a solid-phase hexapeptide ligand library remain to be clarified by more extensive mass spectrometric protein identification. Although use of ProteoMiner for biomarker studies is quite feasible and attractive, more extensive characterization of binding proteins and optimized protocols are required for large-scale biomarker studies.

Figure 1 :
1Overview of protein fractionation by sequential use of 3 different columns to separate plasma proteins. All fractions were subjected to ProteoMiner and 2D-DIGE. carbamidomethyl, variable modifications of oxidation, 1 + , 2 + , and 3 + peptide charge, peptide mass tolerance of 2.0 Da, and use of MS/MS tolerance of 1.0 Da for all tryptic-mass searches.

Figure 2 :
2Overview of the protein contents fractionated by liquid chromatography and ProteoMiner. The fractionated protein samples were loaded onto SDS-PAGE, and the protein contents were visualized by silver staining.depending on their affinity. This fraction contained similar amounts of only 4 major proteins, as revealed by SDS-PAGE(Figure 2), and they may have been absorbed to ProteoMiner in proportion to their original amount.The concentration and amount of protein samples before and after ProteoMiner treatment are summarized in Supplementary

Figure 3 :
3Effects of ProteoMiner treatment were examined by 2D-DIGE. The ProteoMiner-treated and untreated samples were labeled with Cy3 and Cy5, respectively, mixed, and separated by 2D gel electrophoresis. Note that a significant number of protein spots showed different intensities between the 2 samples. The dye-swapped images are shown in Supplementary Figure 2. (a) Original plasma; (b) flow-through fraction of HiTrap Blue HP column; (c) binding fraction of HiTrap Blue HP column. (d) Flow-through fraction of HiTrap Protein G HP column. (e) Binding fraction of HiTrap Protein G HP column; 0 mM fraction. (f) 100 mM fraction. (g) 150 mM. (h) 200 mM. (i) 250 mM. (j) 1 M fraction. (k) Resource Q column.

Figure 4 :
4Localization of protein spots showing different intensities between the ProteoMinor-treated and untreated samples. Panels (a-k) correspond to those in Figure 3. The protein spot numbers corresponds to those in Supplementary Tables 5 and 6. −/−: number of protein spots without ProteoMiner treatment/those with ProteoMiner treatment.

Table 1 :
1List of the identified proteins and their reported concentration.The  table with the references for the protein concentration is shown in Supplementary Table 7 in Supplemrntary material available online at doi: 10.1155/2011/39615.protein spots were then recovered from the gel pieces using an automated spot recovery machine. The recovered protein spots were extensively washed with a solution containing acetonitrile and ammonium bicarbonate minimum and treated with trypsin (Promega, Madison, WI, USA) at 37 • C overnight. The tryptic digests were recovered from the gel pieces, concentrated by vacuum, and resolubilized with 0.1% trifluoroacetic acid. The final tryptic digests were subjected to mass spectrometry, which was performed using the LXQ linear ion trap mass spectrometer (Thermo Electron, San Jose, CA, USA). The Mascot software (version 2.3.0; Matrix Science, London, UK) was used to search for the mass of the peptide ion peaks against the SWISS-PROT database (Homo sapiens, 471472 sequences in Sprot 57.5 fasta file). The search parameters were as follows: trypsin digestion allowing up to 3 missed tryptic cleavages, fixed modifications ofProtein name
Normal concentration
μg/mL

Adiponectin
2-17
Albumin
35000-52000
Alpha-1-antitrypsin
900-2000
Alpha-1B-glycoprotein
150-300
Alpha-2-macroglobulin
1300-3000
Apolipoprotein A-I
1000-2000
Apolipoprotein A-II
190-300
Apolipoprotein A-IV
110-220
Apolipoprotein D
60-90
Apolipoprotein E
30-60
Carboxypeptidase N
30
Ceruloplasmin
190-370
Clusterin
250-420
Coagulation factor X
10
Complement C3
900-1800
Complement C4-A
25-90
Fibrinogen beta chain
520-1420
Fibrinogen gamma chain
490-1340
Ficolin-2
1-12
Ficolin-3
3-54
Haptoglobin
200-2000
Haptoglobin-related protein
32-41
Inter-alpha-trypsin inhibitor
(heavy chain H3)
100-200

Paraoxonase/arylesterase 1
58-61
Prothrombin
100
Serotransferrin
2000-3600
Transthyretin
200-400
Vitronectin
240-530
Zinc-alpha-2-glycoprotein
60-80

AcknowledgmentsThis paper was supported by the Ministry of Health, Labor, and Welfare and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan, T. Kondo was a principal funding recipient. The authors report no conflicts of interests.
Mining the plasma proteome for cancer biomarkers. S M Hanash, S J Pitteri, V M Faca, Nature. 4527187S. M. Hanash, S. J. Pitteri, and V. M. Faca, "Mining the plasma proteome for cancer biomarkers," Nature, vol. 452, no. 7187, pp. 571-579, 2008.

The road from discovery to clinical diagnostics: lessons learned from the first FDA-cleared in vitro diagnostic multivariate index assay of proteomic biomarkers. Z Zhang, D W Chan, Cancer Epidemiology Biomarkers and Prevention. 1912Z. Zhang and D. W. Chan, "The road from discovery to clinical diagnostics: lessons learned from the first FDA-cleared in vitro diagnostic multivariate index assay of proteomic biomarkers," Cancer Epidemiology Biomarkers and Prevention, vol. 19, no. 12, pp. 2995-2999, 2010.

Mass spectrometric detection of tissue proteins in plasma. H Zhang, A Y Liu, P Loriaux, Molecular and Cellular Proteomics. 61H. Zhang, A. Y. Liu, P. Loriaux et al., "Mass spectrometric detection of tissue proteins in plasma," Molecular and Cellular Proteomics, vol. 6, no. 1, pp. 64-71, 2007.

Mining the plasma proteome for disease applications across seven logs of protein abundance. Q Zhang, V Faca, S Hanash, Journal of Proteome Research. 101Q. Zhang, V. Faca, and S. Hanash, "Mining the plasma proteome for disease applications across seven logs of protein abundance," Journal of Proteome Research, vol. 10, no. 1, pp. 46-50, 2011.

Protein equalizer technology : the quest for a "democratic proteome. P G Righetti, E Boschetti, L Lomas, A Citterio, Proteomics. 614P. G. Righetti, E. Boschetti, L. Lomas, and A. Citterio, "Protein equalizer technology : the quest for a "democratic proteome"," Proteomics, vol. 6, no. 14, pp. 3980-3992, 2006.

Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands. V Thulasiraman, S Lin, L Gheorghiu, Electrophoresis. 2618V. Thulasiraman, S. Lin, L. Gheorghiu et al., "Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands," Electrophoresis, vol. 26, no. 18, pp. 3561-3571, 2005.

Prefractionation techniques in proteome analysis: the mining tools of the third millennium. P G Righetti, A Castagna, P Antonioli, E Boschetti, Electrophoresis. 262P. G. Righetti, A. Castagna, P. Antonioli, and E. Boschetti, "Prefractionation techniques in proteome analysis: the mining tools of the third millennium," Electrophoresis, vol. 26, no. 2, pp. 297-319, 2005.

Proteome analysis in the clinical chemistry laboratory: myth or reality?. P G Righetti, A Castagna, F Antonucci, Clinica Chimica Acta. 3572P. G. Righetti, A. Castagna, F. Antonucci et al., "Proteome analysis in the clinical chemistry laboratory: myth or reality?" Clinica Chimica Acta, vol. 357, no. 2, pp. 123-139, 2005.

Plucking, pillaging and plundering proteomes with combinatorial peptide ligand libraries. P G Righetti, E Boschetti, A Zanella, E Fasoli, A Citterio, Journal of Chromatography A. 12176P. G. Righetti, E. Boschetti, A. Zanella, E. Fasoli, and A. Citterio, "Plucking, pillaging and plundering proteomes with combinatorial peptide ligand libraries," Journal of Chromatog- raphy A, vol. 1217, no. 6, pp. 893-900, 2010.

Proteomic analysis of human blood serum using peptide library beads. L Sennels, M Salek, L Lomas, E Boschetti, P G Righetti, J Rappsilber, Journal of Proteome Research. 610L. Sennels, M. Salek, L. Lomas, E. Boschetti, P. G. Righetti, and J. Rappsilber, "Proteomic analysis of human blood serum using peptide library beads," Journal of Proteome Research, vol. 6, no. 10, pp. 4055-4062, 2007.

Comparison of protein enrichment strategies for proteome analysis of plasma. J E Bandow, Proteomics. 107J. E. Bandow, "Comparison of protein enrichment strategies for proteome analysis of plasma," Proteomics, vol. 10, no. 7, pp. 1416-1425, 2010.

Evaluation of the combination of bead technology with SELDI-TOF-MS and 2-D DIGE for detection of plasma proteins. C Sihlbom, I Kanmert, H Von Bahr, P Davidsson, Journal of Proteome Research. 79C. Sihlbom, I. Kanmert, H. Von Bahr, and P. Davidsson, "Evaluation of the combination of bead technology with SELDI-TOF-MS and 2-D DIGE for detection of plasma proteins," Journal of Proteome Research, vol. 7, no. 9, pp. 4191- 4198, 2008.

A proteomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation. E Ernoult, A Bourreau, E Gamelin, C Guette, Journal of Biomedicine & Biotechnology. 2010Article ID 927917, 8 pagesE. Ernoult, A. Bourreau, E. Gamelin, and C. Guette, "A pro- teomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation," Journal of Biomedicine & Biotechnology, vol. 2010, Article ID 927917, 8 pages, 2010.

Deep proteome profiling of sera from never-smoked lung cancer patients. J S K Au, W C S Cho, T T Yip, Biomedicine and Pharmacotherapy. 619J. S. K. Au, W. C. S. Cho, T. T. Yip et al., "Deep proteome profiling of sera from never-smoked lung cancer patients," Biomedicine and Pharmacotherapy, vol. 61, no. 9, pp. 570-577, 2007.

Plasma gelsolin protein: a candidate biomarker for hepatitis B-associated liver cirrhosis identified by proteomic approach. C Marrocco, S Rinalducci, A Mohamadkhani, G M D'amici, L Zolla, Blood Transfusion. 8C. Marrocco, S. Rinalducci, A. Mohamadkhani, G. M. D'Amici, and L. Zolla, "Plasma gelsolin protein: a candidate biomarker for hepatitis B-associated liver cirrhosis identified by proteomic approach," Blood Transfusion, vol. 8, supplement 3, pp. s105-s112, 2010.

Cholangiocarcinoma: update and future perspectives. M Gatto, M C Bragazzi, R Semeraro, Digestive and Liver Disease. 424M. Gatto, M. C. Bragazzi, R. Semeraro et al., "Cholangiocar- cinoma: update and future perspectives," Digestive and Liver Disease, vol. 42, no. 4, pp. 253-260, 2010.

Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics. T Kondo, S Hirohashi, Nature Protocols. 16T. Kondo and S. Hirohashi, "Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics," Nature Protocols, vol. 1, no. 6, pp. 2940-2956, 2007.

Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis. T Okano, T Kondo, T Kakisaka, Proteomics. 613T. Okano, T. Kondo, T. Kakisaka et al., "Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel elec- trophoresis," Proteomics, vol. 6, no. 13, pp. 3938-3948, 2006.

Plasma proteomics of pancreatic cancer patients by multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE): Up-regulation of leucine-rich alpha-2-glycoprotein in pancreatic cancer. T Kakisaka, T Kondo, T Okano, Journal of Chromatography B. 8521-2T. Kakisaka, T. Kondo, T. Okano et al., "Plasma proteomics of pancreatic cancer patients by multi-dimensional liquid chromatography and two-dimensional difference gel elec- trophoresis (2D-DIGE): Up-regulation of leucine-rich alpha- 2-glycoprotein in pancreatic cancer," Journal of Chromatogra- phy B, vol. 852, no. 1-2, pp. 257-267, 2007.

Differences among techniques for highabundant protein depletion. N Zolotarjova, J Martosella, G Nicol, J Bailey, B E Boyes, W C Barrett, Proteomics. 513N. Zolotarjova, J. Martosella, G. Nicol, J. Bailey, B. E. Boyes, and W. C. Barrett, "Differences among techniques for high- abundant protein depletion," Proteomics, vol. 5, no. 13, pp. 3304-3313, 2005.

Assessment of the reproducibility of random hexapeptide peptide library-based protein normalization. R C Dwivedi, O V Krokhin, J P Cortens, J A Wilkins, Journal of Proteome Research. 92R. C. Dwivedi, O. V. Krokhin, J. P. Cortens, and J. A. Wilkins, "Assessment of the reproducibility of random hexapeptide peptide library-based protein normalization," Journal of Pro- teome Research, vol. 9, no. 2, pp. 1144-1149, 2010.

Indepth exploration of cerebrospinal fluid by combining peptide ligand library treatment and label-free protein quantification. E Mouton-Barbosa, F Roux-Dalvai, D Bouyssié, Molecular and Cellular Proteomics. 95E. Mouton-Barbosa, F. Roux-Dalvai, D. Bouyssié et al., "In- depth exploration of cerebrospinal fluid by combining peptide ligand library treatment and label-free protein quantification," Molecular and Cellular Proteomics, vol. 9, no. 5, pp. 1006-1021, 2010.

Combinatorial peptide ligand library plasma treatment: advantages for accessing low-abundance proteins. O Beseme, M Fertin, H Drobecq, P Amouyel, F Pinet, Electrophoresis. 3116O. Beseme, M. Fertin, H. Drobecq, P. Amouyel, and F. Pinet, "Combinatorial peptide ligand library plasma treat- ment: advantages for accessing low-abundance proteins," Electrophoresis, vol. 31, no. 16, pp. 2697-2704, 2010.

A novel serum protein similar to C1q, produced exclusively in adipocytes. P E Scherer, S Williams, M Fogliano, G Baldini, H F Lodish, Journal of Biological Chemistry. 27045P. E. Scherer, S. Williams, M. Fogliano, G. Baldini, and H. F. Lodish, "A novel serum protein similar to C1q, produced exclusively in adipocytes," Journal of Biological Chemistry, vol. 270, no. 45, pp. 26746-26749, 1995.

AdipoQ is a novel adipose-specific gene dysregulated in obesity. E Hu, P Liang, B M Spiegelman, Journal of Biological Chemistry. 27118E. Hu, P. Liang, and B. M. Spiegelman, "AdipoQ is a novel adipose-specific gene dysregulated in obesity," Journal of Biological Chemistry, vol. 271, no. 18, pp. 10697-10703, 1996.

cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1). K Maeda, K Okubo, I Shimomura, T Funahashi, Y Matsuzawa, K Matsubara, Biochemical and Biophysical Research Communications. 2212K. Maeda, K. Okubo, I. Shimomura, T. Funahashi, Y. Mat- suzawa, and K. Matsubara, "cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1)," Biochemical and Biophys- ical Research Communications, vol. 221, no. 2, pp. 286-289, 1996.

Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. Y Nakano, T Tobe, N H Choi-Miura, T Mazda, M Tomita, Journal of Biochemistry. 1204Y. Nakano, T. Tobe, N. H. Choi-Miura, T. Mazda, and M. Tomita, "Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma," Journal of Biochemistry, vol. 120, no. 4, pp. 803-812, 1996.

Adiponectin and metabolic syndrome. Y Matsuzawa, T Funahashi, S Kihara, I Shimomura, Arteriosclerosis, Thrombosis, and Vascular Biology. 241Y. Matsuzawa, T. Funahashi, S. Kihara, and I. Shimo- mura, "Adiponectin and metabolic syndrome," Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 24, no. 1, pp. 29-33, 2004.

Adiponectin and protection against type 2 diabetes mellitus. J Spranger, A Kroke, M Möhlig, The Lancet. 3619353J. Spranger, A. Kroke, M. Möhlig et al., "Adiponectin and protection against type 2 diabetes mellitus," The Lancet, vol. 361, no. 9353, pp. 226-228, 2003.

Low plasma adiponectin levels predict progression of coronary artery calcification. D M Maahs, L G Ogden, G L Kinney, Circulation. 1116D. M. Maahs, L. G. Ogden, G. L. Kinney et al., "Low plasma adiponectin levels predict progression of coronary artery calcification," Circulation, vol. 111, no. 6, pp. 747-753, 2005.

Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. Adults. E E Calle, C Rodriguez, K Walker-Thurmond, M J Thun, New England Journal of Medicine. 34817E. E. Calle, C. Rodriguez, K. Walker-Thurmond, and M. J. Thun, "Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. Adults," New England Journal of Medicine, vol. 348, no. 17, pp. 1625-1638, 2003.

Serum level of adiponectin and the risk of liver cancer development in chronic hepatitis C patients. T Arano, H Nakagawa, R Tateishi, International Journal of Cancer. 1299T. Arano, H. Nakagawa, R. Tateishi et al., "Serum level of adiponectin and the risk of liver cancer development in chronic hepatitis C patients," International Journal of Cancer, vol. 129, no. 9, pp. 2226-2235, 2011.

Associations of insulin resistance and adiponectin with mortality in women with breast cancer. C Duggan, M L Irwin, L Xiao, Journal of Clinical Oncology. 291C. Duggan, M. L. Irwin, L. Xiao et al., "Associations of insulin resistance and adiponectin with mortality in women with breast cancer," Journal of Clinical Oncology, vol. 29, no. 1, pp. 32-39, 2011.

Leptin and adiponectin in pancreatic cancer: connection with diabetes mellitus. T Krechler, M Zeman, M Vecka, Neoplasma. 58T. Krechler, M. Zeman, M. Vecka et al., "Leptin and adiponectin in pancreatic cancer: connection with diabetes mellitus," Neoplasma, vol. 58, pp. 58-64, 2011.

Circulating adiponectin levels and risk of endometrial cancer: the prospective nurses' health study. P T Soliman, X Cui, Q Zhang, S E Hankinson, K H Lu, American Journal of Obstetrics and Gynecology. 2042P. T. Soliman, X. Cui, Q. Zhang, S. E. Hankinson, and K. H. Lu, "Circulating adiponectin levels and risk of endometrial cancer: the prospective nurses' health study," American Journal of Obstetrics and Gynecology, vol. 204, no. 2, pp. 167.e1-167.e5, 2011.

Interaction between adiponectin and leptin influences the risk of colorectal adenoma. T Yamaji, M Iwasaki, S Sasazuki, S Tsugane, Cancer Research. 7013T. Yamaji, M. Iwasaki, S. Sasazuki, and S. Tsugane, "Interaction between adiponectin and leptin influences the risk of colorec- tal adenoma," Cancer Research, vol. 70, no. 13, pp. 5430-5437, 2010.

Prognostic significance of adiponectin levels in non-metastatic colorectal cancer. P Ferroni, R Palmirotta, A Spila, Anticancer Research. 271 BP. Ferroni, R. Palmirotta, A. Spila et al., "Prognostic sig- nificance of adiponectin levels in non-metastatic colorectal cancer," Anticancer Research, vol. 27, no. 1 B, pp. 483-489, 2007.

Mechanisms underlying the anti-proliferative actions of adiponectin in human breast cancer cells, MCF7-dependency on the cAMP/protein Kinase-A pathway. G Li, L Cong, J Gasser, J Zhao, K Chen, F Li, Nutrition and Cancer. 631G. Li, L. Cong, J. Gasser, J. Zhao, K. Chen, and F. Li, "Mecha- nisms underlying the anti-proliferative actions of adiponectin in human breast cancer cells, MCF7-dependency on the cAMP/protein Kinase-A pathway," Nutrition and Cancer, vol. 63, no. 1, pp. 80-88, 2011.

Membrane anchoring and release of carboxypeptidase M: implications for extracellular hydrolysis of peptide hormones. R A Skidgel, G B Mcgwire, X Y Lix, Immunopharmacology. 321-3R. A. Skidgel, G. B. McGwire, and X. Y. Lix, "Membrane anchoring and release of carboxypeptidase M: implications for extracellular hydrolysis of peptide hormones," Immunophar- macology, vol. 32, no. 1-3, pp. 48-52, 1996.

Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1α in the circulation. D A Davis, K E Singer, M De La, Luz Sierra, Blood. 10512D. A. Davis, K. E. Singer, M. De La Luz Sierra et al., "Identi- fication of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1α in the circulation," Blood, vol. 105, no. 12, pp. 4561-4568, 2005.

Combinatorial peptide ligand libraries for urine proteome analysis: investigation of different elution systems. G Candiano, V Dimuccio, M Bruschi, Electrophoresis. 3014G. Candiano, V. Dimuccio, M. Bruschi et al., "Combinatorial peptide ligand libraries for urine proteome analysis: investiga- tion of different elution systems," Electrophoresis, vol. 30, no. 14, pp. 2405-2411, 2009.


Pain originating from the sacroiliac joint is a common non-traumatic musculoskeletal complaint in elite inline-speedskaters -an observational study

Alexander Ruhe
Tino Bos
Arne Herbert
Pain originating from the sacroiliac joint is a common non-traumatic musculoskeletal complaint in elite inline-speedskaters -an observational study
R E S E A R C H Open AccessSpeedskatingNon-traumaticSacro-iliac jointSport-specificPain
Study design: Observational studyObjectives: To investigate common non-traumatic musculoskeletal complaints of the low back in elite inlinespeedskaters of the German national team. Summary of background data: Traumatic injuries associated with falls or collisions are well documented in speedskaters but so far no studies have investigated non-traumatic low back pain. Previously, the sacroiliac joint was suspected as a frequent origin of complaint, we aimed to investigate this assumption.Methods: Two chiropractors examined elite inline-speedskaters of the German national team during three sports events between summer 2010 and 2011. A test cluster of five provocative tests for the sacroiliac joint was selected based on reliability and validity.Results: A total of 37 examinations were conducted on 34 athletes with low back pain during the three sport events. The reported pain intensities ranged from mild to moderate pain (VAS 23.4 ± 13.4 to 35.1 ± 19.2). About 90% of cases showed involvement of the SI joint of which again 90% presented with left sided symptoms. Conclusions: Non-traumatic complaints of the low back originating from the left sacroiliac joint frequently occur in competitive inline speedskaters.

Background

Much has been written about injuries associated with falls or collisions in recreational inline-skating [1][2][3][4][5], but so far no attention has been put on non-traumatic musculoskeletal complaints. In fact, very little research has been conducted at all concerning sport specific complaints of whatever cause in inline-speedskaters.

Inline speedskating is one of the fastest growing disciplines in competitive skating worldwide and races are held in a variety of formats and on a variety of surfaces. The competitive inline speedskating combines a movement pattern similar to speedskating on ice and involves pack-oriented competition modes known from cycling [6]. The competitions are generally held at roller skating rinks with oval tracks of about 100 m in circumference. Races involve the athletes attempting to establish the best time while racing counter-clockwise around the track [7].

In order to prevent future non-traumatic injuries, epidemiologic studies are needed to determine the magnitude of such injuries found within elite in-line speedskaters. Previous general observations by coach and medical staff observed frequent low back complaints which appeared to show SI involvement. The coach of the national speedskating team has expressed an interest in documenting the impact of these injuries in order to investigate this assumption and develop recommendations for rehabilitation and prevention.

We aimed at investigating whether the SI joint is a pain source of speedskaters presenting with low pain. To our knowledge, this is the first study documenting non-traumatic musculoskeletal complaints involving the lumbo-sacral area in inline-speedskaters.

Materials and methods

The involved practitioners were invited by the German inline-speedskating national team to provide chiropractic services for both staff and athletes. Three sport events were included for data collection: 1) European Championships 2010, 2) Annual Cadre Training 2010 and 3) European Championships 2011.

The data collection for this study was conducted prior to any potential therapeutic interventions. Due to the unpredictable onset of the complaints, no protocol could be established for when the examinations were to be conducted (e.g. pre-or post competition). Also, usually no full physical examination was performed as time restraints would often only allow for a focused regional examination.

The locations for the examinations varied both between and during the events. Tests were conducted on-site in the open or in locker rooms. There were no specific procedures as to when and how the athletes were to present to the chiropractor. They did so either at the advice of a member of staff or self-referred at a time of their choosing.

Before the examination of the athletes, consent to participate in this study was obtained. As this was an observational study only and examinations were conducted irrespective of the conduction of this study, no ethics approval was sought. In addition, German regulations do not require ethics approval for this type of research [8]. However, all research was conducted in accordance with the Declaration of Helsinki on Ethical Principles for Medical Research Involving Human Subjects.

Participants

Physical examination findings of all German athletes presenting to the chiropractor with pain in the lumbo-sacral area of non-traumatic origin were included in this study. For the purpose of this study, non-traumatic origin was defined as pain or discomfort not directly associated with a previous traumatic event such as collisions or falls. Athletes were excluded if pain radiated below the gluteal folds or there were other complaints suggesting neurological impairments (e.g. paraesthesia or muscle weakness).

As this study aimed to investigate possible SI involvement irrespective of any previous history of similar or identical complaints, findings of participants that had been examined at an earlier sport event were also included. However, regardless of the number of complaints only the first low back examination per event was to be included.

Procedures

The pain intensity was assessed by means of a 100 mm VAS scale that the athlete filled out prior to the physical examinations.

Based on a literature search we used a protocol to test different aspects of the SI joint consisting of Gaenslen's [9], distraction, thigh thrust, compression and sacral thrust tests as their validity has been previously demonstrated [9][10][11].

At each of the three occasions the orthopedic testing was conducted by an experienced chiropractor (TB and AH) of the German Chiropractic Sports Council. Prior to assessing the symptomatic athletes, all procedures were practiced on 5 individuals to ensure that the involved practitioners were performing the tests in an identical fashion. The inter-or intra-rater reliability was not assessed.

The tests were conducted in the same order each time and bilaterally, starting with the symptomatic side. In order to minimize overstressing of the involved structures and thereby provoking false positives due to repetitive testing, each maneuver was immediately stopped if pain was produced. The chiropractor then proceeded with the next test ( Table 1).

Involvement of the SI joint was assumed if a minimum of three out of five tests were positive on the same side.

The examination findings were documented as either "positive" or "negative" with regards to reproducing familiar pain. A response was noted as "unclear" if the athlete could decide whether a test reproduced pain or not. For the statistical analysis, such a result was counted as a negative response.

Statistical analysis

Simple descriptive statistics were applied for patient demographics and the reported pain intensities. Results were expressed as means, standard deviations (SD) and, where appropriate, 95% confidence intervals (CI).

All statistical analysis was performed with IBM SPSS 19 for Windows (SPSS Inc, Chicago, USA).

Results

Participants

Over the course of 12 months and three events a total of 37 examinations were conducted on 34 athletes. Of these, three speedskaters presented with low back pain at both European Championships.

A total of 27 German athletes participated in the 2010 European Championships. Twelve of these (44%) visited the chiropractor because of low back discomfort. Of the 25 participating speedskaters in the 2010 cadre training camp, eleven (40%) reported lumbo-sacral complaints and participated in this study. During the 2011 European Championships 14 of the 16 participating athletes reported low back pain (88%).

Each participant had been skating competitively for around 5 years. The characteristics of the participants and the reported pain intensities are shown in Table 2.

Four athletes were assessed at two separate events by different examiners. The reported pain intensities ranged from mild to moderate pain [12] (VAS 23.4 ± 13.4 to 35.1 ± 19.2) pre-examination and kept none of the athletes from competing in the events.

Participants primarily described a tension in the lumbo-sacral region or unilateral pain in this area of non-traumatic origin that was not radiating beyond the gluteal folds.

Physical examination findings

All athletes were able to tolerate the examination procedures and there was no trend towards an increased likelihood of positive findings towards the end of the testing sequence.

Apart from a single athlete, all other participants 2010 European Championships with pain in the lumbo-sacral region showed SI involvement (11/12, 92%). In the vast majority of these cases, the left side was involved (9/11, 82%). Of the 11 speedskaters at the Cadre training camp 2010 with a symptomatic low back, two athletes did not show evidence of SI involvement. Where the tests suggested the SI joint as the source of pain (9/11, 82%), in all but two the left side was involved (8/9, 89%). The team for the 2011 European Championships consisted of 16 athletes. Of the 14 patients with pain in the SI area, all but three presented with SI involvement (11/14, 79%). Where the physical examination pointed towards SI involvement, the left side was identified in 91% (10/11) of cases (Table 3).

Overall, of those athletes presenting with unilateral pain in the lumbo-sacral area during these three events, about 90% (31/34, 91%) originated from the SI origin, with 87% (27/31) presenting with left sided involvement. None of the speedskaters described bilateral symptoms or had bilaterally positive examination findings.

Discussion

This study indicates that the SI joints were frequently involved in this cohort of elite speedskaters with nontraumatic low back pain whereby the left side was affected in the vast majority of cases. The study design did also not allow prevalence estimates.

So far there is no universally accepted gold standard for the diagnosis of pain originating from the lumbar spine or sacroiliac (SI) joint but anesthetic or provocative injections have been recommended by some pain Table 1 Procedures for testing the sacroiliac joint Test Description

Distraction

The participant is positioned supine while pressure is applied to the anterior superior iliac spine directed posteriorly and laterally [23].

2 Thigh thrust The participant is lying supine, the hip is flexed to 90°and the knee flexed. A posterior shearing stress is applied along the line of the femur [23].

Compression

The participant is side-lying facing away from the examiner. Downward pressure is applied to the upper most iliac crest [23].

Sacral thrust

The participant is lying prone while three thrusts were applied to the sacrum in an anterior direction [23].

Gaenslen

The participant is asked to lie supine on the edge of a table. The leg being tested is hyperextended at the hip so that it hung over the table. The other leg is flexed at the hip and knee. The participant is instructed to hold the non-tested leg with both arms while the the pelvis is stabilized by the examiner and passive pressure is applied to the tested leg to hold it in the hyperextended position. The examiner then applies additional pressure to place the hip into a position of further extension and adduction. A positive test was noted if pain was provoked or reproduced [24]. physicians [13]. However, these procedures are invasive and expensive, and therefore may not be suitable for routine on-site clinical use. In addition, their validity has been questioned as well [14]. As local anesthetic blocks could not be employed for this study, we relied on orthopedic provocative maneuvers instead. At first sight it appears reasonable to assume that simply stressing a joint should provoke pain of this origin. However, such provocative tests are problematic as it is unlikely that only the intended structure is loaded in isolation. Consequently, if a familiar pain is reproduced by such a stress test, it remains unclear if this is evidence that the targeted structure alone is involved or whether another nearby one that has been stressed simultaneously [14]. If, however, several positive findings of a test cluster were to be combined, greater diagnostic confidence is permitted. While these points have to be kept in mind, diagnostic procedures feasible for on-site clinical application have to be selected in order to establish a diagnosis as a basis for any type of intervention.

After several studies with inconclusive or conflicting results [11], a recent study investigating the reliability of motion-and pain provocation tests for the sacroiliac (SI) joint concluded that a combination of three out of five tests should be employed for acceptable reliability [15]. With regards to validity, Lasett et al. found that performing the distraction, thigh thrust, compression and sacral thrust tests offered the best diagnostic value for SI restrictions. Two positive tests were the best predictors of a positive intra-articular SI block [10]. We used a combination of these recommendations.

Overall, a high percentage of athletes complained about low to medium intensity SI symptoms with the left side being predominately involved. For those studies with negative SI tests, other structures such as muscles, discs, facet joints or other structures of the kinematic chain may be suspected as the pain source. As we did not specifically test for these structures, we are unable to comment on this.

While this points towards an increased biomechanical stress and demand on these structures, the high number of cases presenting with a low pain intensity may be attributed to the convenience of having chiropractic services available on site.

The fact that the majority of athletes participating in the 2011 European Championships presented with SI symptoms does therefore not necessarily point towards a higher number of cases. Instead, the observed trend may simply be attributed to a higher utilization of chiropractic services as athletes got familiar with it.

As the low average pain intensities particularly at this event suggest, athletes may have routinely presented to the chiropractor with only minor discomfort or tension for which they normally may not have sought medical attention. This can be attributed to the fact that even minor feelings of discomfort may adversely affect performance during competition. Consequently, it is difficult to determine from our data the athletes affected by truly clinically relevant SI complaints.

The high percentage of positive SI tests may be surprising considering the reported low pain intensity scores. The examiners took care to challenge the SI joint just sufficiently to reproduce the symptoms. However, while less force/stress may have been required at higher pain intensities, nociceptive input from surrounding structures such as muscles may have also increased the possibility of false positives. In fact, it appears unlikely that the SI joint is solely responsible for symptoms without surrounding structures being involved to some degree [16]. In addition, other factors that we may not have tested for in all athletes, such as disc or spinal pathologies, could have contributed to positive SI-test findings.

In addition, the possibility of false positives cannot be excluded as the sequence of five tests may have stressed the area sufficiently to produce pain. However, as the testing sequence remained identical and no trend towards more positive tests were observed with later tests, it may be assumed that the results remained unaffected by repetitive testing.

While the study design does not allow any concluding on causation, the results nevertheless tempt us to hypothesize that the force vectors associated with counter-clockwise track orientation may play a major role. While ground reaction forces in speed skating remain relatively low, maximal moments at the hip are high and reach values of 140-160 Nm when skating in the straight part of the track [17]. As the athlete is leaning inwards to follow the curves in counter-clockwise direction, even greater values are expected on hips and, in extension, the SI joints. It has been further shown that in ice speedskaters higher loads are applied to the left leg when skating the curves compared to the right leg [18]. This may therefore explain the predominantly left sided symptoms.

Although similar biomechanics apply, it remains unclear whether the findings presented here can also be transferred to speedskating on ice and further studies are needed to investigate this.

Implications for training and rehabilitation

The testing procedures did not allow any conclusion on the causative nature of the SI symptoms. However, routine self-mobilization procedures [19] for the SI joint may be indicated unless the pain is associated with an inflammatory process due to overuse rather than joint hypomobility.

As previously discussed, it cannot be excluded that the other structures such as the sacroiliac or sacrotuberal ligaments contributed to the reported symptoms and positive test findings. However, structures directly surrounding the SI joint will most likely be addressed as well during a therapeutic intervention or rehabilitation as any true isolation of a desired structure appears impossible.

In addition, balancing agonistic and antagonistic lower extremity muscles with regards to length and strength is an important preventive measure as direct and indirect force transmission occurs across the sacrum and ilium [20]. Muscles found to be affected include the iliopsoas, rectus femoris, tensor fascia lata, latissimus dorsi, hip adductors [21], gluteus and quadratus lumborum [16,21], as well as the piriformis muscle [20].

Although maintaining individual muscle flexibility and strength is important, retraining functional coordination between multiple muscle groups is a key to a recovery. This can for example be facilitated by individually targeted lumbo-pelvic stabilization exercises, proprioceptive training as well as sport-specific activities [22].

Strengths and limitations

The cluster consisting of five tests for the SI joints constitutes a major strength of this study with regards to the validity of the results.

Unfortunately, the nature of such field observations as described here entails certain limitations. Although the causality cannot be predicted, the SI joint findings may appear to be a result from overuse associated with training or competitions. It is therefore not feasible to assess such participants in a controlled, non-competitive clinical setting. Further complications are associated with the low number of elite athletes available and the fact that the members of the national team are located all across Germany.

Secondly, due to time restraints between competitions and training sessions, no extended health questionnaires could be distributed nor full physical examinations conducted on all occasions. This may have resulted in bias as previous injuries or treatments may have affected the results of this study. However, the athletes were asymptomatic prior to the competitions as they otherwise would not have been on the team. This indicates that the presenting complaints most likely occurred in association with practice or races at the respective event.

Thirdly, some athletes may have visited the chiropractors immediately after symptoms were noticed, whereas others would have arrived later on. Also, some of the speedskaters were examined right after an exhausting competition that may have initiated the symptoms (with all implications regarding fatigue, muscle tone etc.) whereas others would have rested between training sessions or competitions. This means that physical examinations were not performed under the same overall conditions which in turn may have affected the results for both pain intensity and examination findings. However, this is what the sports physician has to accommodate with, including limited time available for the examinations and treatments that have to be scheduled around competitions and training. Although desirable, these time restraints also prohibited a broader, more detailed musculoskeletal examination cluster to be conducted with each athlete that would have allowed commenting on other biomechanical deficits.

In addition, a high risk of selection bias was unavoidable, e.g. with regards to the small group of athletes, which rendered prevalence estimates problematic. Despite all intentions to test objectively, it is also possible that the involved chiropractors may have been biased towards detecting positive SI-joint tests as previous experience suggested such findings. However, it may be concluded nevertheless that the left SI joint was commonly involved in those presenting with low back pain.

In order to reduce the risk of bias, future studies may want to conduct more detailed examinations including spine and lower extremities. This would allow commenting on other potential pain generating structures that may further contribute to positive SI-joint findings. Examiners otherwise not involved in this sport and blinded to our results may also be beneficial. However, as mentioned before, such time consuming procedures may not be feasible during competitive events. In addition, it may be difficult to gather such elite athletes in a more controlled setting and test them during practice sessions where a reduced willingness to perform may not elicit similar results.

Conclusion

Non-traumatic pain or discomfort in the lumbo-sacral region was commonly encountered in this group of elite inline speedskaters. In the vast majority of the participants, the SI joint appears to be the origin of the complaint, of which the left side is involved in nearly 90% of cases. We hypothesize that this may be associated with increased loading on this leg when skating the curves.

Table 2
2Participant characteristics and pain intensitySport event

Table 3
3Physical examination findingsAthlete No.
Side involved
Provocative tests
Number of positive tests

Distraction
Thigh Thrust
Sacral thrust
Gaenslen

European Championships 2010

01
Left
-
-
+
+
+
3

02
Left
+
+
-
+
-
3

03
Right
+
+
+
+
4

04
Left
+
+
+
+
+
5

05
Left
-
-
-
+
unclear
(1)

06
Left
+
+
-
+
-
3

07
Left
+
+
+
+
+
5

08
Right
+
+
+
3

09
Left
+
+
+
+
4

10
Left
+
-
+
-
+
3

11
Left
+
+
+
+
4

12
Left
-
+
+
+
-
3

Cadre training camp 2010

13
Left
+
+
+
-
3

14
Left
-
-
+
-
-
(1)

15
Left
+
+
+
+
4

16
Right
+
+
-
+
-
3

17
Left
+
+
+
+
4

18
Left
+
+
+
-
-
3

19
Left
-
+
-
+
+
3

20
Left
-
-
+
-
-
(1)

21
Left
+
+
unclear
+
+
4

22
Left
+
+
+
3

23
Left
+
+
-
+
-
3

European Championships 2011

24
Left
+
+
-
+
-
3

25
Left
+
-
-
+
+
3

26
Left
-
-
+
-
-
(1)

27
Right
+
+
+
+
+
5

28
Left
+
+
+
-
-
3

29
Left
-
+
+
+
-
3

30
Left
-
+
-
+
+
3

31
Left
-
-
-
-
-
(0)

32
Left
-
-
+
+
+
3

33
Left
+
+
+
+
+
5

34
Right
-
-
-
-
+
(1)

35
Left
+
+
+
+
+
5

36
Left
+
+
-
+
-
3

37
Left
+
+
-
+
-
3

-:negative,+ : positive, n = number

Ruhe et al. Chiropractic & Manual Therapies 2012, 20:5
http://chiromt.com/content/20/1/5

Ruhe et al. Chiropractic & Manual Therapies 2012, 20:5 http://chiromt.com/content/20/1/5
AcknowledgementsWe thank the coaches and athletes for consenting to participate in this study, as well as Christin Maskus for assisting in the documentation of examination findings. The authors would also like to acknowledge René Fejer MHSc (Clin Biomech), PhD for his helpful advice during the process of drafting the manuscript.Authors' contributions AR conducted the statistical analysis and drafted the manuscript. TB and AH conducted the physical examinations and assisted in drafting the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
In-line skating injuries. Epidemiology and recommendations for prevention. R A Schieber, C M Branche-Dorsey, Sports Med. 19427Schieber RA, Branche-Dorsey CM: In-line skating injuries. Epidemiology and recommendations for prevention. Sports Med 1995, 19:427.

Risk factors for injuries from in-line skating and the effectiveness of safety gear. R A Schieber, C M Branche-Dorsey, G W Ryan, G W Rutherford, J A Stevens, O' Neil, J , N Engl J Med. 335Schieber RA, Branche-Dorsey CM, Ryan GW, Rutherford GW, Stevens JA, O'Neil J: Risk factors for injuries from in-line skating and the effectiveness of safety gear. N Engl J Med 1996, 335:1630-1635.

Inline skating. Patterns of injury and risk group. R E Hilgert, Mhr Dallek, K H Jungbluth, Der Unfallchirurg. 101Hilgert RE, Dallek MHR, Jungbluth KH: Inline skating. Patterns of injury and risk group. Der Unfallchirurg 1998, 101:845-850.

Injuries associated with inline skating in the European region. S Mulder, A Hutten, Accid Anal Prev. 34Mulder S, Hutten A: Injuries associated with inline skating in the European region. Accid Anal Prev 2002, 34:65-70.

Dental injuries in inline skatinglevel of information and prevention. D Fasciglione, R Persic, Y Pohl, A Filippi, Dent Traumatol. 23Fasciglione D, Persic R, Pohl Y, Filippi A: Dental injuries in inline skating - level of information and prevention. Dent Traumatol 2007, 23:143-148.

Characteristics of inline speedskating-incremental tests and effect of drafting. A Krieg, T Meyer, S Clas, W Kindermann, Int J Sports Med. 27Krieg A, Meyer T, Clas S, Kindermann W: Characteristics of inline speedskating-incremental tests and effect of drafting. Int J Sports Med 2006, 27:818-823.

. CIC General Régulations. Fédération Internationale de Roller Sports. CIC General Régulations. Fédération Internationale de Roller Sports; 2011.

C Fetzer, C H Gleiter, Nicht-interventionelle Studien in der klinischen Forschung: Qualität gewinnt. Deutsche Zeitschrift für Klinische Forschung. Fetzer C, Gleiter CH: Nicht-interventionelle Studien in der klinischen Forschung: Qualität gewinnt. Deutsche Zeitschrift für Klinische Forschung 2011, 7/8:41-44.

Diagnostic validity of criteria for sacroiliac joint pain: a systematic review. K M Szadek, P Van Der Wurff, M W Van Tulder, W W Zuurmond, R S Perez, J Pain. 10Szadek KM, van der Wurff P, van Tulder MW, Zuurmond WW, Perez RS: Diagnostic validity of criteria for sacroiliac joint pain: a systematic review. J Pain 2009, 10:354-368.

Diagnosis of sacroiliac joint pain: validity of individual provocation tests and composites of tests. M Laslett, C N Aprill, B Mcdonald, S B Young, Man Ther. 10Laslett M, Aprill CN, McDonald B, Young SB: Diagnosis of sacroiliac joint pain: validity of individual provocation tests and composites of tests. Man Ther 2005, 10:207-218.

Clinical tests of the sacroiliac joint. A systematic methodological review. Part 1: Reliability. P Van Der Wurff, R H Hagmeijer, W Meyne, Man Ther. 5van der Wurff P, Hagmeijer RH, Meyne W: Clinical tests of the sacroiliac joint. A systematic methodological review. Part 1: Reliability. Man Ther 2000, 5:30-36.

The visual analogue pain intensity scale: what is moderate pain in millimetres? Pain. S L Collins, R A Moore, H J Mcquay, 72Collins SL, Moore RA, McQuay HJ: The visual analogue pain intensity scale: what is moderate pain in millimetres? Pain 1997, 72:95-97.

Evaluation of sacroiliac joint interventions: a systematic appraisal of the literature. M P Rupert, M Lee, L Manchikanti, S Datta, S P Cohen, Pain Physician. 12Rupert MP, Lee M, Manchikanti L, Datta S, Cohen SP: Evaluation of sacroiliac joint interventions: a systematic appraisal of the literature. Pain Physician 2009, 12:399-418.

Provocative sacroiliac joint maneuvers and sacroiliac joint block are unreliable for diagnosing sacroiliac joint pain. J M Berthelot, J J Labat, Le Goff, B Gouin, F Maugars, Y , Joint Bone Spine. 73Berthelot JM, Labat JJ, Le Goff B, Gouin F, Maugars Y: Provocative sacroiliac joint maneuvers and sacroiliac joint block are unreliable for diagnosing sacroiliac joint pain. Joint Bone Spine 2006, 73:17-23.

The reliability of selected motion-and pain provocation tests for the sacroiliac joint. H S Robinson, J I Brox, R Robinson, E Bjelland, S Solem, T Telje, Man Ther. 12Robinson HS, Brox JI, Robinson R, Bjelland E, Solem S, Telje T: The reliability of selected motion-and pain provocation tests for the sacroiliac joint. Man Ther 2007, 12:72-79.

Sacroiliac Joint Involvement in Activation of the Porcine Spinal and Gluteal Musculature. A Indahl, A Kaigle, O Reikerås, S E Holm, J Spinal Disord. 12Indahl A, Kaigle A, Reikerås O, Holm SE: Sacroiliac Joint Involvement in Activation of the Porcine Spinal and Gluteal Musculature. J Spinal Disord 1999, 12:325-330.

Coordination of leg muscles during speed skating. J J De Koning, G De Groot, G J Van Ingen Schenau, J Biomech. 24de Koning JJ, de Groot G, van Ingen Schenau GJ: Coordination of leg muscles during speed skating. J Biomech 1991, 24:137-146.

Speed Skating the Curves: a study of muscle coordination and power production. J J De Koning, G De Groot, G J Van Ingen Schenau, J Appl Biomech. 7de Koning JJ, de Groot G, van Ingen Schenau GJ: Speed Skating the Curves: a study of muscle coordination and power production. J Appl Biomech 1991, 7:344-358.

Mechanics and treatment of the sacroiliac joint. R L Dontigny, J Manipulative Physiol Ther. 1DonTigny RL: Mechanics and treatment of the sacroiliac joint. J Manipulative Physiol Ther 1993, 1:3-12.

Sacroiliac joint pain: practical management. H Prather, Clin J Sport Med. 13Prather H: Sacroiliac joint pain: practical management. Clin J Sport Med 2003, 13:252-255.

Load application to the sacrotuberous ligament: Influences on sacroiliac joint mechanics. A Vleeming, J P Van Wingerden, C J Snijders, R Stoeckart, T Stijnen, Clin Biomech. 4Vleeming A, Van Wingerden JP, Snijders CJ, Stoeckart R, Stijnen T: Load application to the sacrotuberous ligament: Influences on sacroiliac joint mechanics. Clin Biomech (Bristol, Avon) 1989, 4:204-209.

Low Back Disorders: Evidence-Based Prevention and Rehabilitation. 2 edition. Human Kinetics. S M Mcgill, McGill SM: Low Back Disorders: Evidence-Based Prevention and Rehabilitation. 2 edition. Human Kinetics; 2007.

Validity and reliability of clinical tests for the sacroiliac joint. P Cattley, J Winyard, J Trevaskis, S Eaton, Australasian Chiropractic & Osteopathy. 10Cattley P, Winyard J, Trevaskis J, Eaton S: Validity and reliability of clinical tests for the sacroiliac joint. Australasian Chiropractic & Osteopathy 2002, 10:73-80.

Praxis der konservativen Orthopädie Stuttgart: Thieme Verlag. H-P Bischoff, J Heisel, H: Orthopädische Locher, Untersuchung, 10.1186/2045-709X-20-5Bischoff H-P, Heisel J, Locher H: Orthopädische Untersuchung. Praxis der konservativen Orthopädie Stuttgart: Thieme Verlag; 2005, 13-15. doi:10.1186/2045-709X-20-5

Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Ruhe, Chiropractic & Manual Therapies. 205Pain originating from the sacroiliac joint is a common non-traumatic musculoskeletal complaint in elite inlinespeedskaters -an observational study. Submit your manuscript at www.biomedcentral.com/submitCite this article as: Ruhe et al.: Pain originating from the sacroiliac joint is a common non-traumatic musculoskeletal complaint in elite inline- speedskaters -an observational study. Chiropractic & Manual Therapies 2012 20:5. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

. Ruhe, Chiropractic & Manual Therapies. 205Ruhe et al. Chiropractic & Manual Therapies 2012, 20:5