the objective of biomarker discovery is to identify specific protein markers susceptible to improve early diagnosis survey therapeutic outcomes and facilitate the development of novel drug candidates [ 1 , 2 ] . the fundamental approach is based on the assumption that the pathology of concern will affect some physiological processes causing changes in the protein expression levels . proteins generating similar signals in both sample groups are ignored while significantly up- and downregulated proteins become potential biomarkers . differential expression profiling requires both a sensitive technology to discern any tiny differences and a high - throughput system in order to process large series of samples required to reach statistical significance . protein differential display techniques such as two - dimensional gel electrophoresis ( 2-de ) , one- or two - dimensional liquid chromatographic ( lc - ms ) , or surface - enhanced laser desorption / ionization time of flight mass spectrometry ( seldi - tof - ms ) are regarded as the most powerful tools for establishing fingerprint profiles [ 36 ] . many reports regarding the application of the seldi - tof - ms technology have been published since its introduction in 1993   and its first use for disease detection . one of the key features of seldi - tof - ms is its ability to provide rapid protein expression profiles from a variety of biological samples with minimal requirements for purification and separation of proteins prior to mass spectrometry . seldi - tof - ms profiling studies revealed that biological fluids contain many proteins with low molecular weight ( < 15  kda ) not resolved on conventional 2d gels [ 6 , 9 ] . as can be seen in figure 1 , the seldi technique consists in surface arrays involving various chromatographic models based on both classic chemistries ( normal phase , hydrophobic , cation- and anion - exchange surfaces ) and specifically affinity - coated surfaces ( immobilized metal affinity capture : imac ) . after the binding phase of the sample to these surfaces , the unbound proteins are washed out while retained molecules are overlaid with an energy - absorbing matrix . in the final step , mass spectra are recorded using a laser for the ionization and a tof mass spectrometer for its resolving power . recent interest in the field has yielded a large number of candidate biomarkers in various diseases [ 1035 ] . however , the small size and poor design of some studies drove validation of these biomarkers quite challenging [ 3641 ] .   in the context of clinical proteomic using seldi - tof - ms , many recent reviews discussed newly identified disease biomarkers [ 13 , 21 , 22 , 24 , 27 , 30 , 35 , 4244 ] . the present review focuses on technical challenges encountered with the seldi - tof - ms technology taking into account new insights coming from the last three years . critical steps that should be undertaken to avoid any bias , to maximize reproducibility and detection sensitivity , with the final aim to find relevant , specific , and robust biomarkers are addressed [ 45 , 46 ] . for prospective studies , current knowledge on the different biological fluid sources available for seldi - tof - ms experiments a successful biomarker research program starts with a careful study design and the preparation of a detailed protocol . many manuscripts report encountered problems , emphasizing the importance of standard operating procedures ( sops ) , clinical protocols , instrument tuning , and stabilization [ 3740 , 4763 ] . only critical points will be discussed in this review . in the early phase of biomarker discovery , the clinical question addressed has to be defined in the disease(s ) context collecting adequate control samples . indeed , it can be criticized that in many published studies , patients were compared to healthy subjects rather than to patients presenting similar diseases or clinical signs . the avoidance of bias is not trivial and must be addressed throughout the whole study , from its design to the data analysis and interpretation ( cf . figure 2 ) . current proteomics and genomics technologies are extremely sensitive and can detect very small changes in expression levels . some of these changes may arise from biological differences related to disease or pharmacological treatment . they could also result from the heterogeneity of the patient panels tested across multiples sites , the inherent biological complexity , and the diversity of sample types . small differences in sample collection , processing , and analytical techniques could have some impact on the outcomes of the study . as a consequence , clinical data may be site- , study- , population- , or sample - dependent , without any actual clinical relevance [ 53 , 62 , 6466 ] . the key factor for maximizing reproducibility in biomarker research is to identify and minimize all potential sources of preanalytical and analytical bias [ 53 , 55 ] ( table 1 ) . adherence to strict guidelines and sops is critical to reach the highest operating standards for data quality and reproducibility [ 37 , 38 , 47 , 50 , 51 , 53 , 55 , 62 , 63 , 67 ] . sops also facilitate the validation of biomarkers by other groups using different sets of samples . besides the instrumentation and the methodologies related to chromatography - mass spectrometry analysis , the nature , quality , and number of clinical samples to process are key elements to be considered for any proteomic approaches . in order to provide positive answer to any precise clinical question , the investigator has to make a choice among the most relevant biological target samples ( body fluid , tissue , etc . ) . many criteria must be considered at this level , that is , availability , easiness of collection , stability , composition , proximity with disease location , patient discomfort , ethics , and so forth .    for biomarker discovery using seldi - tof - ms , a great variety of   biological sample types can be used ; major applications concern biofluids ( plasma , serum , urine , saliva , cerebrospinal liquid , bronchoalveolar wash out , nipple aspirate fluid , tears , amniotic fluid ) [ 1 , 1014 , 18 , 19 , 21 , 22 , 24 , 28 , 42 , 48 , 49 , 6877 ] , and tissues or cell extracts [ 78 , 79 ] . among them , serum , plasma , urine , and saliva are the most popular . many variables related to collection , storage , and conditions for sample preparations have to be carefully considered . these parameters have been commented in literature according to the nature of the biofluids [ 48 , 49 , 54 , 57 , 61 , 67 , 73 , 74 , 8084 ] . in this section of the review , advantages and drawbacks of the most popular fluids are presented in table 2 as well as formulated recommendations . when reviewing a series of studies [ 4749 , 51 , 54 , 57 , 58 , 60 , 61 , 67 , 68 , 73 , 8183 , 85 ] general guidelines can be forwarded . after clot formation , samples can be transported or stored on wet ice for 3 hours before centrifugation . anticoagulant edta is preferred and its processing should be realized as soon as possible after sampling , ideally within the first hour . although storage at low temperature promotes peptides and proteins stability , one should not recommend storing plasma samples at 4c due to the cold activation of platelets . prior any freezing , plasma can be depleted in platelets by using a filtration step ; aliquots are then freezed at   80c .   according to the  hupo ppp specimens committee recommendations , plasma appears preferable to serum because it contains less peptides of degradation and consequently presents less variability [ 57 , 81 , 86 ] . in order to avoid the presence of platelet related peptides , the authors also recommend to use platelet - poor plasma obtained by centrifugation followed by a filtration step . however , the choice of serum could be justified when studying diseases related to coagulation abnormalities . a controversial parameter is the addition of protease inhibitors ( pis ) to the samples . some authors found that the addition of a pi cocktail induces significant differences in protein profiles when compared to crude samples [ 58 , 83 ] . whenever directly introduced during phlebotomy , pi allows fluid stabilization for at least 2 hours at room temperature by reducing proteolysis damages . however , pi presents some additional drawbacks such as the presence of highly concentrated components in the cocktail which can compete later on for protein array interactions . another important factor to decrease the risk of variation , bias , and errors is the communication between researchers and medical staff . one generally considers that 70% of the errors are due to human intervention ( mostly due to communication problems ) while only 30% appear instrumental related errors . the mode of specimen collection ( veni - puncture or arterial puncture ) , the site of collection , the position of the patient , or the tourniquet technique can influence the concentration of certain blood constituents . it is generally advised to discard those kinds of samples , but when the disease studied involves spontaneous hemolysis , this can not reasonably be done . this mode of collection has the advantage that only few drops of blood are needed ( particularly interesting for neonatal and repeated screening ) . moreover , it does not require specific medical support for sampling , which could be promising for multiple collects realized by the patient at home , in the perspective of a treatment follow - up , for instance . however , like blood specimen , such factors have to be taken into consideration to improve reproducibility of sample collection . saliva protein composition varies with circadian rhythm , diet , age , gender , and physiological status . it is also affected by the method of sample collection ( stimulated versus non - stimulated saliva production ) . food ingestion increases the proteolysis activity and then collection before lunch rather than after is recommended . the addition of pi can reduce but not completely eliminate the impact of the proteolysis . it will stabilize , qualitatively and quantitatively , the saliva proteome for up to 48 hours . regarding storage conditions , it is preferred to store the saliva specimens at   80c rather than at   20c where the preservation of the protein content could not be guaranteed for more than 1 month . interestingly , repeated freeze - thaw cycles ( 4/5 ) do not seem to significantly alter saliva protein profile . it is mainly an aqueous solution ( 95% of water ) of waste electrolytes and metabolites , organic components ( urea , uric acid ) , and proteins at low concentrations in healthy individuals ( 150  mg / day ) . urine proteome variation depends mainly on plasma composition due to its role as blood content regulator and on the integrity of the glomerular filtration step leading to a large intra and intersubjects variability . protein and salt concentrations can vary along the day for a same subject ( first void compared to midstream urine samples ) [ 80 , 88 ] . progressive degradation of urine proteome due to proteolytic activity can be prevented by pi addition only up to 2 hours of storage . as already mentioned for blood and saliva , up to 5  freeze / thaw cycles do not significantly affect urine proteome profile . other fluids such cerebrospinal fluid , nipple aspirates , tears , synovial fluid , bronchoalveolar lavage , follicular , and amniotic fluids have already been explored by seldi - tof - ms [ 15 , 20 , 33 , 34 , 69 , 75 , 89 , 90 ] . these fluids are generally used to study well - localized diseases . despite the presence in such fluids of some plasma proteins , their implication to study systemic diseases is not recommended and difficult to apply in routine diagnosis due to risk and discomfort related to collection . one of the most challenging aspects in studying body fluids protein profiles remains the detection of the deep proteome . the protein concentration dynamic range detectable by means of maldi - tof or seldi - tof - ms is about 2 orders of magnitude , whereas the range in blood reaches about 10 orders of magnitude [ 91 , 92 ] . as protein binding onto chromatographic surface depends on its affinity , its concentration , but also on the surface binding capacity , one can imagine that the competition between different proteins for binding sites is very complex . a highly abundant protein with low affinity for the chip surface and a low abundant protein with high affinity may give similar peak intensities in the final seldi mass spectrum . several fractionation procedures are now available to decrease the sample protein concentration dynamic range [ 85 , 93102 ] . a major inconvenience for sample fractionation is the resulting low sample throughput capacity , due to a significant increase of the duration of analysis and to a risk of poor reproducibility affecting data treatment . additionally the same proteins can be presented in different fractions challenging the comparison of their abundance between samples . several methods have been proposed for fractionation such as centrifugal ultrafiltration , precipitation by organic solvents , electrophoresis , chromatography ( on - column or on - magnetic beads ) , or subcellular localization . the choice will be made based on the nature of the sample to be analysed and the protein properties ( molecular weight , localization , abundance , etc . ) . all these sample preparation methods have already been discussed by other reviewers [ 85 , 94 , 101 , 103 , 104 ] . recently , with the growing interest in studying posttranslational modifications new methodologies set up to isolate rare amino acid - containing peptides ( cys , met , trp , his ) or ptm peptides ( phosphopeptides , glycopeptides ) have been developed . one of the most widely used approach for highly abundant proteins removal in serum and plasma is their depletion using antibodies . despite the depletion of the nine most abundant proteins from serum or plasma samples , this sensitivity problem is most probably inherent to the too low concentration of the peptidome constituents . moreover , some of the abundant proteins act as carrier explaining the codepletion of almost 3000 species ( peptides and proteins ) as observed by several groups . it implied a solid - phase combinatorial library of hexapeptides on which millions of copies of a unique ligand are graft on a bead . this technique , enabling the dilution of abundant protein by rapid saturation of its ligand , concentrates components of the deep proteome which could not reach saturation . this method presents the advantage to reduce the dynamic range between the most and less abundant proteins and peptides . it has also been showed that despite compression of the dynamic range , this technology used for differential studies was only applicable for proteins or peptides which do not reach saturation ( low and medium abundance proteins ) . many studies conducted on different types of samples report good reproducibility and important gain in the number of low abundant species by comparison with analyses performed on corresponding crude samples [ 96 , 98 , 99 , 108111 ] , which make this approach very promising to investigate the deep proteome . the choice of the protein chip array chemistry and the nature of the matrix depend on whether the application requires general profiling or requires a specific protein assay . different array types and binding conditions may generate complementary protein profiles for the same sample . the use of relevant quality controls ( qcs ) is highly recommended and even mandatory in such applications [ 3740 , 4763 ] . qcs should be well - characterized pools of samples processed alongside the experimental samples in order to monitor instrument performances , optimize mass spectrometry settings ( laser energy , etc . ) , compare target protein profiles to those of historical reference samples , and to calculate coefficient of variation for peak intensities as a measure of reproducibility . it is important to point that the resolution and mass accuracy provided by this kind of instrument are rather low compared to high - resolution mass spectrometers ( i.e. , q - tof , ft - ms , etc . ) . using seldi - tof - ms , one could not expect to accurately determined m / z values or peak intensities on complex mixtures . this means that only large differences in peak intensities are to be considered and that peaks of interest have to be identified with more accurate mass spectrometers . beside those instrumental weaknesses , on the contrary to other mass spectrometers , seldi - tof - ms can be used for high throughput analysis . during seldi settings , several events , such as matrix crystallization , ion suppression , and in - source decay occurring during mass spectra acquisition strongly influence the peak intensities . differences in reagents , handling of material , room temperature , and level of humidity may all influence the ( co)crystallization step of matrix molecule with sample causing interday fluctuation . these parameters must be highly controlled and standardized for each study protocol . during the crystallization process easily embedded proteins will be present at higher concentrations in the matrix and consequently more efficiently desorbed and ionized . to improve sample - to - sample reproducibility of maldi ion yield and to increase the precision of peptide quantification , some authors use nitrocellulose in order to improve the homogeneity of the matrix / analyte crystallization [ 55 , 112 , 113 ] . depending on sample composition , ion suppression is another factor that significantly contributes to the variability observed in seldi - tof - ms spectra [ 47 , 50 , 55 ] . indeed , during ionization , if a protonated analyte collides with an unprotonated one which has higher gas - phase basicity , it may pass its proton to the collision partner . this phenomenon is called  ion suppression effect .  in a complex protein mixture like serum , where highly abundant proteins constitute a large proportion of the total protein content , it is possible that such peaks override signals from low abundant peptides . this phenomenon , obviously difficult to prevent in complex samples , would be more easily controlled on mixtures issued from fractionation . another source of variations is the fragmentation of proteins or peptides during mass spectrometric process . fragmentation occurring before the first field - free region is called in - source decay ( isd ) ; it is responsible for consecutive series of ions . showed that isd generates quite additional spectral peaks in the spectrum of proteins contained in serum samples when compared with the data collected for pure reference proteins . one obviously creates isd favourable conditions when optimizing the analytical conditions by maximizing the total peak count , particularly when using a high laser beam which would increase the thermal ions energy and consequently the number of collisions between ions . developed a method which deconvolutes the spectrum by appropriately associating peaks belonging to the same protein . to take benefit of this procedure , common mechanisms accounting for the arising of multiple peaks in mass spectra include , for example , the formation of salt adducts and multiply charged ions . chemical reactions using energy from the laser may take place between sample protein molecules , matrix molecules , or molecules from the washing buffers generating intermolecular complexes known as the formation of these complexes increases the number of spectrum peaks causing artefacts ( i.e. , satellite peak at   + 206 da corresponds to a spa adduct ) . moreover , the performance of the seldi - tof - ms may change over time due to possible fluctuation in the laser intensity and/or detector sensitivity . all these difficulties can be addressed only by substantial reduction in sample complexity and the application of a rigorous standardization program of the entire analytical process . this involves optimized acquisition protocols ( i.e. , avoiding too high laser intensity ) , a fully operational and calibrated instrument and the use of suitable qc samples , similar in nature and complexity to the studied samples . the data preprocessing ( calibration , baseline correction , normalization , peak detection , and peak alignment ) represents a key step for seldi analysis [ 115117 ] . spectra are generally normalized in order to equalize or minimize differential effect due to external variation [ 59 , 115 , 116 , 118 , 119 ] . the widely used total ion current ( tic ) gives a clear indication of the impacts of technical variables such as laser and detector performances , matrix application , and sample amounts . tic normalisation relies on the assumption that the technical parameters are mostly responsible for the largest differences observed between samples . they suggest to examine whether normalisation factors vary systematically between study groups and they recommend to specify the applied methodology ( local or global normalisation , matrix signal excluded or not ) . the ideal normalisation procedure would be to resort to some internal spiking method . one important aspect in seldi - tof - ms data analysis is to avoid false discovery of protein peaks , for which the discriminative power results from random variation . a general criticism concerns the use of inadequate algorithms for data analysis and the problem issued from over - adjustment in combination of high - dimensional data with a low number of cases . those artefacts could be prevented by analysing a sufficient number of samples , by resorting to overfitting - resistant algorithms , by an appropriate validation of the resultant model , and by using optimal spectra processing techniques ( calibration , exclusion of spectral regions affected by high noise , peak alignment , and normalization ) . two others remarks can be formulated from literature reports : ( 1 ) multiple biomarkers have generally a better predictive value than individual markers , and ( 2 ) positive - predictive values of peptide patterns are often insufficient to be recognized as early markers when they concern low - frequency diseases in the population [ 38 , 53 ] . the most commonly used bioinformatics approaches are decision tree - based ones and support vector machines [ 120 , 121 ] . authors generally emphasized on the need for validated model selection using cross validation loop and permutation testing to develop generalized classifier able to correctly predict classification of new samples . identification of candidate biomarkers , while not strictly necessary for diagnostic purpose , can be regarded as extremely satisfying in helping to data interpretation and better understanding the disease . as often criticized , the seldi - tof - ms technology does not provide peptide / protein identification . in order to succeed in the identification by sequencing ( q - tof , tof - tof , ion - trap , etc . ) or peptide fingerprinting ( maldi - tof ) , enrichment and purification of the biomarker of interest is often needed , which is laborious and time consuming . to solve in part this weakness , new proteinchip interface coupled to tandem mass spectrometer was recently developed allowing direct sequencing of peptides   < 6000  da . in all cases , identifications must be corroborated using antibody - based detection ( i.e. , western blot or elisa ) or antibody pull - down with subsequent detection by seldi - tof - ms . it should be noted that the concentration range of widely used biomarkers in plasma samples is remarkably wide and differ from the high milligram until low nanogram per liter range . for example , serum albumin , within a normal concentration range of 3550  mg / ml , is measured as an indication of severe liver disease   or malnutrition , whereas il-6 normally varies in a range of 05  pg / ml , is measured as a sensitive indicator of inflammation or infection .   until now , most of the markers identified after an seldi - tof - ms study could not yet be considered as very specific of a given disease but they are rather representative of disease 's consequences like inflammation or immune response . the most frequently identified proteins so far are haptoglobulin , transthyretin , apolipoproteins , serum amyloid , or complement factors present at g / ml to mg / ml [ 13 , 19 , 2325 , 28 , 38 ] . although individual acute - phase reactions proteins are not satisfactory diagnosis biomarkers , their combined use with other serum biomarkers may enable more sensitive and specific diagnosis ( cf . , figure 3 ) . this phenomenon has recently been termed  host response protein amplification cascade  . the moderate specificity of seldi - discovered biomarkers could be explained by its low sensibility . to date , seldi - tof - ms has not yet identified any protein marker present at ng / ml level . this probably indicates that the lowest detect limit of this technology is around g / ml as considered by diamandis . to overcome this limited detection sensitivity , the serum ( or plasma ) proteins fractions could then be loaded on different arrays using complementary binding conditions . moreover , the decisive advantage of the mass spectrometry technologies is the capacity to detect protein variants , protein fragments , and posttranscriptional modifications ( ptms ) , which is usually not possible with affinity - based technologies . it is now recognized that those components may be disease - specific and can be considered as potential biomarkers ( i.e. , modified transthyretin forms in ovarian cancer in figure 4 and in familial amyloidotic polyneuropathy ) [ 123 , 129 , 130 ] .   in the last two years , lots of applications using seldi - tof - ms were published for diagnostic of cancers [ 25 , 42 , 44 , 131 ] , especially breast [ 10 , 17 ] , prostate [ 21 , 132 , 133 ] , and colorectal cancer . other recent papers concerning infectious diseases , neurodegenerative disorders , renal diseases [ 26 , 134 ] , and chronic inflammatory diseases [ 19 , 135 ] also demonstrated the great potential of the technique . seldi - tof - ms technology was also used to predict response to therapy , particularly in cancers . rcken and whelan described in detail the use of seldi - tof - ms to not only predict responses to cancer therapy but also demonstrate its interest in the follow - up of metastasis disease progression and in the development of drug resistance [ 44 , 136 ] . recently platelet factor 4 ( pf4 ) appeared to be a biomarker for infliximab nonresponse in crohn 's disease and rheumatoid arthritis [ 29 , 137 ] .    for most of these studies , a validation phase should assess the validity of the described potential biomarkers against a larger and more heterogeneous population of patients . the robustness of the candidate markers has to be tested against a level of biological variability that more accurately reflects the variability in the target population . unfortunately , several groups failed to validate the biomarker discovered in their pilot study , such as mclerran et al . they concluded that their first study samples most likely had biases in the sample selection . another validation performed by engwegen et al . using distinct patient populations confirmed that saa peak clusters are associated to renal cell carcinoma . such examples demonstrate the importance to strictly control parameters such as storage , clotting , time of analysis , instrument performances , sample selection , and statistical classification method . the urgent need for sops in clinical proteomics research is therefore absolutely mandatory reflecting a growing trend in the field [ 19 , 53 , 62 , 63 ] . interaction between researchers , clinicians , and statisticians is also a key element for the success . altogether , these applications of seldi - tof - ms technology illustrate its capability for discrimination and follow - up of a multitude of diseases using different body fluids as well as certain therapeutic response prediction . it is worth mentioning that fda approved recently the first diagnostic tool ( named ova1 ) issued from seldi proteomic researches . taking into account herein and previously described recommendations , seldi - tof - ms offers very exciting opportunities to discover not only diagnostic but also prognostic and mechanistic markers for a number of major diseases . to face the general criticism , standardized procedures and recommendations to minimize bias however , some challenges still remain , as for all other proteomic approaches , due in part to the complexity and the wide dynamic range of the samples . sample fractionation and/or enrichment procedure , such as peptide ligand affinity beads , will certainly be the solution to visualise the deep proteome . in addition , improvements in mass spectrometry instrumental performances could be expected ( higher resolution , reducing adduct formation , and ion suppression ) , contributing further to more reliable and faster biomarkers discovery .
protein profiling using seldi - tof - ms has gained over the past few years an increasing interest in the field of biomarker discovery . 
 the technology presents great potential if some parameters , such as sample handling , seldi settings , and data analysis , are strictly controlled . 
 practical considerations to set up a robust and sensitive strategy for biomarker discovery are presented . 
 this paper also reviews biological fluids generally available including a description of their peculiar properties and the preanalytical challenges inherent to sample collection and storage . 
 finally , some new insights for biomarker identification and validation challenges are provided .