diabetes has become a global epidemic , 
 and its patient population 
 will increase drastically in the coming years , according to the international 
 diabetes federation . despite its clinical diagnosis using fasting 
 plasma glucose ( fpg ) and glycated hemoglobin a ( hba1c ) assays and home monitoring using blood glucose meters , 
 one of the major challenges in diabetes management is the longitudinal 
 monitoring of its progression and therapeutic responses . glucose meters 
 measure the transient blood glucose levels in the plasma , while hba1c 
 assays measure the average level of hba glycation inside the red blood 
 cells for the preceding 23 months . the 
 glycation gap , defined as the difference between the measured hba1c 
 and the hba1c value predicted from glycated serum proteins , has been 
 associated with microvascular complications of diabetes . therefore , efforts are ongoing to incorporate glycated albumin ( ga ) 
 in the plasma , i.e. , glycated human serum albumin ( hsa ) , as an additional 
 clinical marker for the average blood glucose level over a period 
 of 23 weeks . on the other hand , a variety of platforms and sample preparation protocols are utilized 
 to measure glucose , hba1c , and ga , separately , each using different 
 methods based on liquid chromatography , immunoassay , electrochemistry , 
 electrophoresis , etc . consequently , it is so far not 
 a routine practice to perform parallel analysis of these markers under 
 the same clinical settings and integrate the results in a timely manner . a unified platform that could concurrently measure multiple classes 
 of diabetes markers , including but not limited to glucose , hba1c , 
 and ga , and encompass multiple time scales ( e.g. , transient , days , 
 weeks , and months ) of individual glycemia would make major contributions 
 to diabetes theranostics and management . because the proteome 
 reflects an individual s physiopathological 
 states at a given time , proteomics is a powerful tool for diagnosing 
 disease and monitoring progression and therapeutic responses . the 
 majority of current clinical protein assays rely on the enzyme - linked 
 immunosorbent assay ( elisa ) , which has the advantage of high sensitivity 
 and ease of operation . however , elisa suffers several significant 
 limitations : ( a ) multiplexing greater than approximately 10 antibodies 
 is difficult due to the cross - reactivity of antibodies ; ( b ) antibodies 
 are not available for the vast majority of proteins , particularly 
 for their modified isoforms ; and ( c ) assay development is lengthy 
 and expensive . in contrast , mass spectrometry ( ms ) allows rapid and 
 multivariate analysis of complex patterns of biomarkers without having 
 specific antibodies available . however , the penetration 
 of ms - based proteomics into the in vitro diagnostics 
 market has remained low . ms - based platform 
 has to achieve the ( 1 ) sensitivity , ( 2 ) throughput , and ( 3 ) robustness , 
 comparable to or even better than those of elisa , in order to find 
 wider clinical acceptance . the focus of clinical proteomics has been 
 on analyzing low - abundance proteins using bottom - up proteomics ( i.e. , 
 analysis of proteolytic peptides ) , which faces the challenge 
 of the huge dynamic range in biological fluids such as blood and urine 
 and the difficulty of identifying all protein isoforms ( or proteoforms ) , including splicing , modifications , cleavages , 
 etc . , and quantifying their stoichiometry . there have been recent 
 advances in top - down proteomics , i.e. , large - scale identification 
 and characterization of full - length proteins , but its clinical potentials remain largely unexplored . mass spectrometry is making inroads into clinical diagnostics , which 
 creates opportunities for new and improved assays . in this work , we describe a new nanoflow liquid chromatography  mass 
 spectrometry ( lc  ms ) assay , enabled by our silicon - microfluidic - chip 
 platform , the multinozzle emitter array chip ( mea chip ) , for rapid and multidimensional monitoring of diabetes , through direct 
 top - down proteomics analysis of submicroliter volumes of human blood 
 samples . the single - plex mea chips were designed using the l - edit 
 software ( v15 , tanner research ) . however , the new design 
 contained a three - layer si  si  glass structure that monolithically 
 integrated several functional modules on a single chip ( figure 1a ) . specifically , the electrospray emitters were 
 constructed between the two silicon layers , while all other functional 
 components ( including the lc and trap columns ) were built between 
 the glass and silicon layers . a through - hole in the middle silicon 
 layer was produced to connect emitters with lc channels . the silicon 
 layers offer the ease for fabricating complex structures , while the 
 glass cover provides the imaging window for real - time monitoring of 
 on - chip processes such as the bead packing . mea chips were examined 
 by light microscope and scanning electron microscope ( sem ) to confirm 
 integrity of each component . for this work , the lc column was designed 
 to be 5 cm ( length )  100 m ( width )  100 m 
 ( depth ) , and the trap column was 1 cm ( length )  300 m 
 ( width )  120 m ( depth ) . the microfabricated emitter had 
 nozzles with a cross - section of 25 m  25 m and 
 a protruding length of 120 m . the extraction segment was designed 
 for analyte enrichment but was not utilized in this work . ( a ) schematics of the chip with 
 the top - view ( i ) and cross - sectional view ( ii ) . all other 
 functional components including a trap column ( green ) and a lc column 
 ( red ) were produced between the silicon and glass layers . ( b ) high - resolution photographs showing the 
 chip and its assembly with a custom - built manifold and fittings , relative 
 to a u.s . the dimensions of the lc column were 5 cm ( length ) 
  100 m ( width )  100 m ( depth ) , and the trap 
 column was 1 cm ( length )  300 m ( width )  120 m 
 ( depth ) . the microfabricated emitter had nozzles with a cross - section 
 of 25 m  25 m and a protruding length of 120 m . to establish robust fluidic connections 
 for high - pressure on - chip 
 and online nanolc separation , we built a manifold to mechanically 
 assemble the mea chip with capillary tubing connected to the outside 
 nanoflow source ( figure 1b ) . the chip was sandwiched 
 between a peek clamping plate and an aluminum plate and tightly clamped 
 by screws with o - rings in - between to prevent the fluid leakage . the 
 top peek plate had four threaded ports for upchurch fittings to provide 
 connections with capillary tubing . the assembly was then fastened 
 to a translational stage , using a screw in the aluminum plate . no fluid leakage was observed for the mea chip assembly for the flow 
 under a pressure of over 2000 psi in our lc  ms runs . for on - chip lc columns , a 3 m frit was implemented between 
 the lc channel and nozzles to retain beads . lc and trap channels were 
 packed with magic - c4 5 m beads ( pore size of 300  , bruker - michrom ) 
 using an in - house column packing station . briefly , beads were suspended 
 in methanol and sonicated to form a solution of monodispersed particles . then the slurry of particles was forced into the channels on the chip 
 through the sample input holes by a pressurized ( > 1000 psi ) helium 
 gas tank . the pressure gauge was shut off in 20 min , and the system 
 was slowly depressurized for about 1 h before switching to the atmosphere 
 pressure . finally , the backend of the packed channels were sealed by fabricating 
 sol  gel frits to prevent beads 
 from retreating during the lc runs . the sol gel solution was 
 prepared by mixing 34 l of kasil 1 potassium silicate ( pq corp . ) 
 with 6 l of formamide ( sigma ) , followed by vortexing and centrifuging 
 for 1 min . gel solution was 
 introduced to a chip reservoir and then flowed into the channel for 
 2 min . the chip was then incubated on 
 a hot plate at 80 c for over 6 h. after the frit was completely 
 polymerized , the columns with sol the quality and reproducibility of frit fabrication and 
 bead packing were confirmed by microscopic examination , followed by 
 backpressure monitoring for the lc channels under a constant flow 
 rate ( e.g. , 1 l / min ) . ms analysis of standard proteins mixtures . 
 since our on - chip channels were built on inert silicon substrate , 
 we followed the standard protocols for conventional cartridge columns 
 to regenerate lc and trap channels . pure hba1c and hba0 ( ifcc 
 reference material ) were obtained from 
 lee biosolutions ( st . louis , mo ) . . 34650 , level 14 ) was obtained from bio - rad ( hercules , 
 ca ) . all other chemicals and biologics were obtained from sigma - aldrich 
 ( st . louis , mo ) . for esi - ms response curve of hba1c / hba , calibrator 
 solutions were prepared by mixing pure hba0 and hba1c solutions in 
 lc solvent a ( 5/95 acetonitrile ( acn)/h2o with 0.2% formic 
 acid ( fa ) ) . a set of calibrators with five levels of hba1c ( 0 , 1.9% , 
 5.7% , 10.7% , and 16.7% ) were prepared in triplicate , and each replicate 
 was analyzed at least 3 times . right before use , they were thawed , incubated 
 at 37 c for 10 min , and then diluted 1:1000 in solvent a for 
 lc  ms analysis . for lc ms response curve of glucose / d-(+ ) glucose-6,6-d2 ( hereafter : 
 glucose - d2 ) , calibrator solutions were 
 prepared by mixing pure glucose and glucose - d2 solutions and subequently spiked in the pooled plasma that 
 was diluted 1:100 in solvent a. the concentration of glucose - d2 in the final solution was fixed at 50 m . a set of calibrators with five concentrations of glucose ( 0 , 20 , 50 , 
 100 , and 250 m ) were prepared in triplicate , and each replicate 
 was analyzed at least 3 times . fresh whole blood from both healthy 
 control donors ( n = 8) and type 2 diabetes ( t2d ) 
 patients ( n = 8) 
 and frozen pooled plasma were obtained from innovative research inc . the fresh blood samples were collected in the presence 
 of edta and shipped on ice by fedex to newomics inc . within 23 
 days after collection . aliquots of whole blood samples were stored 
 at 4 c upon arrival and analyzed within 25 days of receipt 
 in order to collect the data described in this work . a 5 l 
 aliquot of each whole blood sample was diluted with 10 l of 
 1 pbs buffer , and afterward the mixture was centrifuged at a 
 speed of 3,000 g for 5 min at room temperature ( rt ) . a 5 l aliquot of the supernatant was reconstituted in 36.7 
 l of lc solvent a and subsequently centrifuged at 14,000 g for 5 min at rt to remove any cellular debris . a total 
 of 35 l of supernatant was collected and stored as the plasma 
 portion , with a final concentration of 1:25 dilution of the beginning 
 whole blood sample . the cell pellet derived from the first centrifuge 
 step was washed with 1 pbs buffer at rt three times and then 
 incubated with 50 l of 1 pbs for 2 h at 37 c to 
 remove the labile pre - hba1c ( schiff base , also called aldimine ) . the cells were subsequently lyzed by suspending 
 the cell pellet in 25 l of hplc - grade water and vortexing for 
 5 min at rt . the hemolysate was then constituted in 470 l of 
 lc solvent a and centrifuged at 14,000 g for 5 min . a total of 450 l of supernatant was collected and stored as 
 the hemolysate portion , with a final concentration of 1:100 dilution 
 of the beginning whole blood sample . finally , an artificial mixture 
 of a 40 l solution was generated by mixing 10 l of 200 
 m glucose - d2 standards , 10 l 
 of its plasma portion ( 1:25 dilution ) , 0.4 l of its hemolysate 
 portion ( 1:100 dilution ) , and 19.6 l of lc solvent a. a 4 l 
 aliquot of the mixture , representing 0.1 l of each whole blood 
 sample , was injected for lc  ms analysis to generate the data 
 shown in figure 2 . samples were prepared in 
 triplicate , and each replicate was analyzed at least 3 times . a top - down - proteomics - centric 
 assay using small volumes of blood 
 samples . ms peaks 
 for free glucose , hsa , hba , and apoa - i , respectively . the representative 
 mass spectra of glucose and different isoforms of hsa , hba , and apoa - i 
 after maxent 1 deconvolution are shown in inserts a the identified protein modifications include glycation , cysteinylation , 
 nitrosylation , oxidation , and truncation . a capillary liquid 
 chromatography system ( caplc ) ( waters corp . ) a volume of 4 l of the processed 
 mixture ( see above ) was injected through an autosampler into the on - chip 
 trap column with a flow rate of 20 l / min . the on - chip lc column 
 was run at a flow rate of 600 nl / min . the solvent a consisted of 5/95 
 acn / h2o with 0.2% fa , and solvent b consisted of 95/5 acn / h2o with 0.2% fa . the lc gradient started at 1% b and was hold 
 at 1% b for 3 min . starting at 3 min , it was linearly increased to 
 20% b in 5 min and then ramped up again to 50% in 42 min . after that , 
 it was increased to 95% b in 5 min and finally returned to the initial 
 condition ( 1% b ) in another 5 min . ms detection was performed using 
 a hybrid quadrupole / orthogonal q - tof api us mass spectrometer ( waters 
 corp . ) with the same ms and ms / ms settings as we described before . the capillary voltage was set to be 3.2 kv , and cone voltage was 
 40 v. nanoelectrospray process on mea emitters was visualized and 
 monitored using a waters nanoflow camera kit equipped with the mlh-10x 
 zoom lenses ( computar ) . ms 
 data were processed 
 using the masslynx 4.0 software package provided with the q - tof instrument . 
 extracted ion chromatograms ( eic ) for all target proteins were generated 
 using their corresponding ions of the charge state at the maximum 
 intensity . the entire peak region in the eic for each protein was 
 summed to acquire their integrated mass spectra . the integrated mass 
 spectrum ( m / z 7501350 for 
 hba and apoa - i , m / z 11001400 
 for hsa ) was then deconvoluted onto a mass scale using the maximum 
 entropy - based algorithm ( maxent 1 ) in masslynx 4.0 . the parameters 
 for maxent 1 were chosen as the following : mass range 12,00018,000 
 da for hba , 63,00069,000 da for hsa , and 24,00032,000 
 da for apoa - i ; resolution 0.10.2 da / channel . the uniform gaussian 
 peak width at the half - height for each protein was determined using 
 its highest intensity peak . the left and right minimum intensity ratio 
 was set to be 40% for hba , 85% for hsa , and 80% for apoa - i . finally , 
 the maxent 1 deconvoluted spectrum was baseline - subtracted with a 
 25-order polynomial , smoothed ( 2  6 da savitzky - golay ) , and 
 centered ( centroid top 80% ) with areas created . the ion intensities 
 in the centered spectra , which employed the corresponding peak areas , 
 were used to quantify each protein isoform . for hba1c , for hsa and 
 apoa - i isoforms , we assumed similar ms responses for unmodified proteins 
 and their different adducts ( e.g. , hsa - cys and hsa - glyc ) in this work . hba1c value was calculated using the ratio 
 between the peak area ( i ) of the charge deconvoluted 
 peak of hba--glyc ( mass , 16029 da ) and those of all prominent 
 hba- isoforms including hba- ( mass , 15867 da ) , hba--sno 
 ( mass , 15897 da ) , and hba--glyc and normalized by the esi - ms 
 response factor determined from the slope of the response curve in 
 figure 3a . the equation is , where i represents the 
 peak area of the charge - deconvoluted peaks , and 0.9516 is the response 
 factor . validation of a top - down - proteomics - centric assay for diabetes 
 monitoring . ( a ) calibration curve for the ratio of hba1c : hba determined 
 by our assay for known molar ratios of the mixtures of purified hba1c 
 and hba0 . ( b ) comparison of hba1c values for the lyphochek hemoglobin 
 a1c linearity set samples ( lot 34650 ) , obtained by our mea chip - based 
 assay (  ) , and the corresponding target values using other commercial 
 assays ( ngsp (  ) and ifcc (  ) ) provided by bio - rad . ( c , 
 d ) comparison of hba1c values for blood samples from t2d patients 
 ( n = 8) , each measured by our mea chip and a commercial 
 tosoh g7 hplc analyzer , respectively , showing the correlation between 
 the values obtained by the two methods ( pearson s correlation , r = 0.9695 , p < 0.0001 , n = 8) ( c ) and the bland - altman plot of the difference between the 
 two methods . the lines were plotted indicating the bias ( 0.80% ) and 
 the upper and lower limits of agreement ( loa ) ( bias 2  
 sd ) ( d ) . similarly , the relative level 
 of hba - sno was calculated as the relative level of glycated 
 albumin ( ga ) was calculated as we 
 used the double weighting of the doubly glycated form of hsa 
 to account for two glycations per hsa . the relative 
 level of hsa - cys was calculated asthe relative 
 level of apoa - i glycation ( gapoa - i ) 
 was calculated as the denominator contained all identifiable apoa - i peaks and 
 represented 
 the total apoa - i . the relative level of methionine oxidation 
 of apoa - i ( apoa - i meto ) 
 was calculated as the percentage of maximum methionine oxidation capacity 
 of apoa - i , in which all apoa - i molecules are modified by 3 methionine 
 sulfoxides . the intensity of unoxidized , 
 singly , doubly , and triply oxidized apoa - i were multiplied by 0 , 1/3 , 
 2/3 , and 1 , respectively , and then summed to obtain the weighted intensity 
 of the oxidized apoa - i . hence , apoa - i meto was obtained by normalizing the weighted intensity 
 of oxidized apoa - i with that of the total apoa - i as for statistical analyses , 
 we utilized the graphpad prism 6 ( graphpad 
 software inc . , la jolla , ca ) . for error bars at each data point , at 
 least triplicate experiments ( n a bland - altman plot was 
 used for comparison between our assay using a mea chip and the commercial 
 hba1c assay using a tosoh g7 instrument . student s t test was performed to examine the differences between 
 controls and t2d patients for hba1c , ga , gapoa - i , hsa - cys , hba - sno , 
 and apoa - i meto , respectively . the p values of the t test were determined by using the unpaired and two - tailed 
 parametric tests without assuming equal variance in both groups . pearson s 
 correlation was performed to investigate the following relationships : 
 ( 1 ) between any two of hba1c , ga , and gapoa - i ; ( 2 ) between hsa - cys 
 and hba1c , ga , and gapoa - i , respectively ; and ( 3 ) between age and 
 hsa - cys , hba - sno , and apoa - i meto , respectively . pearson s 
 coefficient ( r ) and p value ( two - tailed ) were calculated . we developed a mea chip 
 for top - down - proteomics - centric analysis 
 of small volumes of blood samples . figure 1b shows an assembly of our one - plex mea chip ( chip itself is shown 
 in the insert ) used for this study . ms analyses of free glucose and abundant 
 blood proteins relevant to hyperglycemia in diabetes including hemoglobin 
 a ( hba ) , human serum albumin ( hsa ) , and apolipoprotein a - i ( apoa - i ) 
 ( figure 2 ) . using glucose - d2 as the internal standard , we could accurately determine 
 the free glucose concentrations in the blood using the ratio between 
 the lc ms peak areas of the na+ adducts of glucose and glucose - d2 ( m / z 203 
 and 205 , respectively ) ( supplementary figure 1 ) . because glucose has a low hydrophobicity , it was eluted very early 
 in our current 1-h lc gradient on the c4 column however , we were still able 
 to obtain the response curve and quantify glucose from the lc  ms 
 chromatograms . since glucose degrades quickly in the whole blood and 
 needs to be measured immediately after the blood draw , we did not 
 quantify the glucose level in individual whole blood samples in this 
 work . in the future , we could also analyze glucose in a separate column 
 packed with hilic ( e.g. , nh2 ) beads on a 24-plex mea chip 
 if necessary . we next showed quantitation of various isoforms of hsa , 
 hba , and apoa - i in a pooled plasma sample or a fresh whole blood sample 
 from a healthy individual . ms analysis 
 of standard protein mixtures to validate the performance of the on - chip 
 c4 column on our mea chip ( supplementary figure 
 2 ) . we then analyzed diluted blood samples corresponding to 
 a mere 0.1 l of the starting plasma using our mea chip . by 
 comparing the lc ms chromatograms , ms spectra , charge states , 
 and maxent 1 transformed charge - deconvoluted peaks ( inserts ) ( figure 2 ) , we identified various modifications ( cysteinylation , 
 glycation , nitroslylation , methionine oxidation , etc . ) of these proteins 
 in the blood , consistent with the earlier published works . specifically , for hsa , we detected its multiple species , including 
 unmodified , cysteinylated ( at cys34 ) , glycated ( dominantly at lys525 ) , 
 both cysteinylated and glycated , and doubly glycated isoforms ; for 
 hba , we identified glycation at both  and  chains , and 
 nitrosylation at  chain ( at cys93 ) ; for apoa - i , we identified 
 glycation ( hereafter : gapoa - i ) , oxidation at 13 methionine 
 residues ( hereafter : apoa - i meto ) , etc . protein nitrosylation and 
 cysteinylation at cysteine residues are important for signal transduction 
 in response to oxidative stress , while 
 glycation of hba ( and hsa ) is a known marker for glucose metabolism 
 and widely used for diabetes diagnosis . we next developed and 
 validated our assay for diabetes monitoring . 
 using hba1c ( hba glycated at the n - terminus of  chain ) as the 
 example , we worked out the lc ms calibration curve for the 
 hba1c / hba ratio and obtained a response factor of 0.9516 ( figure 3a ) . based on this response curve , we determined 
 the hba1c values for the bio - rad lyphochek hemoglobin a1c linearity 
 set , which contains standard samples to check linearity and verify 
 calibration of commercial instruments for hba1c assays ( figure 3b ) . we plotted the target values of hba1c for different 
 platforms provided in the bio - rad technical datasheet along with the 
 values determined by our mea chip platform . as expected , the values 
 for national glycohemoglobin standardization program ( ngsp)-based 
 methods were grouped higher than those for international federation 
 of clinical chemistry and laboratory medicine ( ifcc)-based methods 
 ( typically + 1.52.0% ) [ note : ifcc% was used 
 in the plot as provided in the datasheet ; however , ifcc values are 
 now generally reported in mmol / mol ] . ngsp - based methods measure the 
 percentage of hba1c in total hba at the protein level , while ifcc - based 
 methods measure the ratio between glycated and nonglycated hexapeptides 
 of hba at the peptide level after enzymatic digestions . the values from our lc  ms 
 assay were consistent with these target values . therefore , our nanoflow 
 lc  ms assay using a mea chip is suitable for quantifying protein 
 glycation as glycemia markers for diabetes . we further confirmed that 
 the hba1c values determined by our assay were consistent with those 
 obtained using conventional methods . as shown in figure 3c and d , our results for the 8 blood samples from t2d patients 
 agreed very well with those obtained by the commercial tosoh g7 hplc 
 platform ( ngsp method ) ( figure 3c : pearson s 
 correlation , r = 0.9695 , p < 
 0.0001 , two - tailed ; and figure 3d : bland - altman 
 plot , limit of agreement ( loa ) , p < 0.0500 ) . we then demonstrated proof - of - principle applications of our assay 
 for monitoring individual glycemia . we analyzed fresh blood samples 
 from a total of 16 individuals ( 8 healthy controls ; 8 type 2 diabetes , 
 t2d ) . we compared the values of hba1c , ga , and gapoa - i between controls 
 and t2d ( figure 4a  c ) . the mean values 
 were significantly higher in t2d than in controls , with 8.44% and 
 5.95% for hba1c , 30.93% and 17.05% for ga , and 5.10% and 3.63% for 
 gapoa - i , respectively . further , we could completely segregate controls 
 from t2d using these markers ( student s t test , p = 0.0090 , 0.0260 , 0.0151 for hba1c , ga , and gapoa - i , respectively ) . we next evaluated the relationship between each two of these three 
 markers for the total 16 samples analyzed ( figure 4d  f ) . all correlated strongly to each other , with r = 0.9268 , 0.8857 , and 0.8495 ( pearson s correlation , p < 0.0001 , two - tailed ) , between hba1c and ga , ga and 
 gapoa - i , and gapoa - i and hba1c , respectively . on the basis of the 
 average in vivo lifetime of their unmodified proteins , 
 hba1c , ga , gapoa - i could manifest the average blood glucose level 
 over a period of 23 months , 23 weeks , and 12 
 days , respectively . therefore , the degree 
 of mutual correlations matches the degree of difference in time scales 
 that these markers represent . ( a , 
 b , c ) quantitation of the glycation levels of hba ( hba1c ) , hsa ( ga ) , 
 and apoa - i ( gapoa - i ) in controls and type 2 diabetes ( t2d ) patients , 
 respectively ( n = 8 for each group ) . the mean hba1c , 
 ga , and gapoa - i levels in t2d were 8.44% , 30.93% , and 5.10% , respectively , 
 while in controls they were 5.95% , 17.05% , and 3.63% , respectively . the p values were calculated using the two - tailed 
 student s t test . ( d , e , f ) correlation between 
 any two values of hba1c , ga , and gapoa - i , respectively , for each individual 
 monitored in ( a  c ) ( pearson s correlation , p < 0.0001 , n = 16 ) . finally , we showed that our assay could provide additional 
 information 
 about oxidative stress and cardiovascular risks for individuals . there 
 is a strong interplay between oxidative stress and diabetes , and the 
 most severe consequences of diabetes are cardiovascular diseases including 
 atherosclerosis . as already shown in 
 figure 2 , our lc ms assay concurrently 
 detected hsa - cys , hba - sno , and apoa - i meto , well - known protein markers 
 for oxidative stress in plasma , hypoxic vasodilation , and oxidative 
 status of high - density lipoprotein ( hdl ) , respectively . we compared the values of these markers for controls 
 and t2d ( figure 5a  c ) . we observed a 
 significant increase of hsa - cys in t2d compared to controls ( mean 
 = 49.59% vs 36.96% , p = 0.0033 , student s t test ) , a nonsignificant decrease of hba - sno ( mean = 20.17% 
 vs 19.33% , p = 0.1489 , student s t test ) , and a nonsignificant increase of apoa - i meto ( mean = 12.01% 
 vs 12.87% , p = 0.1212 , student s t test ) . these results suggested the higher oxidative stress ( via 
 hsa - cys ) and the possible perturbation of vasodilation functions ( via 
 gapoa - i and hba - sno ) in t2d patients . although we observed a significant 
 increase of apoa - i glycation in t2d ( figure 4c ) , the nonsignificant change in the corresponding apoa - i meto ( figure 5c ) awaits further studies . we observed 
 some nonsignificant correlations between hsa - cys and blood glycemia 
 markers hba1c , ga , and gapoa - i , with the values for pearson s 
 correlation ( two - tailed ) , r = 0.4139 , p = 0.1110 ; r = 0.4292 , p = 0.0971 ; 
 and r = 0.3110 , p = 0.2410 for ga , 
 hba1c , and gapoa - i , respectively ( figure 5d ) . we plotted the values of hsa - cys , hba - sno , and apoa - i meto 
 against the ages available for the 8 t2d patients ( figure 5e ) . interestingly , we observed a significant positive 
 correlation between the age and hsa - cys ( pearson s correlation , r = 0.7087 , p = 0.0491 , two - tailed ) , suggesting 
 the increased oxidative stress during aging , which is consistent with 
 the free - radical theory of aging . however , we did not observe a significant 
 correlation between the age and hba - sno or between the age and apoa - i 
 meto . there have been conflicting results about the role and regulation 
 of methionine oxidation of apoa - i in diabetes . future in vitro and in vivo studies will clarify 
 the issue and provide new biological insights into the anti - atherosclerosis 
 functions of hdl . application of the assay for concurrently 
 monitoring individual 
 oxidative stress and cardiovascular risks . ( a , b , c ) comparison of 
 the levels of hsa cysteinylation ( hsa - cys ) , hemoglobin s - nitrosylation 
 ( hba - sno ) , and apoa - i oxidation ( apoa - i meto ) , between controls and 
 t2d patients , respectively ( n = 8 for each group ) . the mean hsa - cys , hba - sno , and apoa - i meto levels in t2d were 49.59% , 
 19.33% , and 12.87% , respectively , while in controls they were 36.96% , 
 20.17% , and 12.01% , respectively . the p values were 
 calculated using the two - tailed student s t test . ( d ) correlation between the levels of hsa - cys and ga ( blue 
  ) , hba1c ( green  ) , and gapoa - i ( red  ) , respectively , 
 for each individual monitored in panels a ( e ) correlation between the 
 age of individual t2d patients and their levels of hsa - cys ( blue  ) , 
 hba - sno ( green  ) , and apoa - i meto ( red  ) , respectively 
 ( pearson s correlation , n = 8) . in this 
 study , we have developed a novel assay for rapid and multidimensional 
 monitoring of diabetes starting from a drop of blood ( figure 6 ) . the key novelty of our assay lies in the combined 
 analysis of small molecules , proteins , and protein post - translational 
 modifications with common pathophysiological themes ( high blood glucose 
 and oxidative stress ) that play important roles in diabetes , through 
 a single lc we have considered several important issues during our assay 
 development : a scheme for rapid and multidimensional monitoring of 
 diabetes 
 using a drop of blood . our top - down - proteomics - centric assay , enabled 
 by our mea chip platform , concurrently measures markers for multitime - scale 
 glycemia ( glucose , gapoa - i , ga , and hba1c ) , oxidative stress ( hsa - cys ) , 
 and cardiovascular risks ( hba - sno , gapoa - i , and apoa - i meto ) in multiple 
 compartments of blood , thereby contributing to better long - term monitoring 
 and disease management of diabetes . the size of various components 
 in the blood drop was not drawn to scale . we assumed 
 similar ms responses for unmodified and different adducts of hsa and 
 apoa - i , respectively . this was mainly due to the fact that unlike 
 hba1c , highly purified and singly modified species of hsa and apoa - i 
 were not available to us . we obtained a response factor of 0.9516 
 for hba1c , which is very close to 1.0 . given that both apoa - i and 
 hsa are bigger in size than hba- , one could presume that the 
 effect of modifications ( e.g. , glycation ) on ms would be bigger for 
 hba- than for hsa and apoa - i . however , even if the response 
 factor were not close to 1.0 for hsa and apoa - i , our assumption would 
 not change our conclusions in a substantial way . first , we were interested 
 in relative changes between normal and patient groups , using the percentage 
 of each species . second , our hsa and apoa - i results were consistent 
 with those of hba1c . third , similar assumptions ( i.e. , response factor 
 = 1.0 ) were made in earlier published works . finally , the main goal of this proof - of - principle work is to demonstrate 
 one of the first top - down - proteomics - centric assays for clinical applications . met - ox is prominent in esi analysis 
 of peptides because met residues are completely exposed to the reactive 
 oxygen species ( ros ) in the solution throughout the experiments . for 
 proteins , met - ox had been observed when the protein was acid - denatured 
 prior to esi analysis using high voltages . in this case , hba was acid - denatured first , which resulted in the 
 complete exposure of met residues in the  chain , for oxidation 
 during esi at 3.5 kv . therefore , multiple met - ox species were observed 
 for hba  chain . however , under our lc  ms conditions , 
 we did not observe a significant population of met - ox species for 
 hba  chain from the fresh whole blood , suggesting that the 
 artificial contribution of mex - ox during our esi process was low . for the previous top - down ms analyses of apoa - i using the similar 
 quantitation methods , samples had to go through a multistep sample 
 preparation procedure including the immunoaffinity capture , wash , 
 and elution prior to ms analysis . in 
 contrast , our one - step sample preparation included only a dilution 
 step for apoa - i in the plasma fraction and therefore would further 
 minimize the artificial met - ox formation during sample preparation . 
 for apoa - i meto calculation , we followed the concept of maximum methionine 
 oxidation capacity by using the weighted oxidation of apoa - i in the 
 numerator . however , we included all identifiable 
 apoa - i peaks in the denominator as the total apoa - i in order to be 
 consistent with that in gapoa - i calculation . similar to calibrations for hba1c values obtained from 
 individual ngsp and ifcc platforms , each of the glycemia values determined 
 by our assay might need calibrations in order to match those already 
 adopted for clinical classification of nondiabetics and diabetics . 
 for example , the current who cutoff value for hba1c is 6.5% . a calibration 
 factor could be applied to our data set to match this value . dynamic range is a challenging issue for both bottom - up 
 proteomics and top - down proteomics . it does not require 
 complex sample preparation such as immunoaffinity enrichment and therefore 
 is not constrained by the dynamic range issue that has long plagued 
 other assays for analyzing low - abundance proteins . however , our mea 
 chip does contain an extraction segment that could be used for enrichment 
 of additional low - abundance species for diabetes monitoring if needed . 
 for conventional microbore / nanobore lc  ms systems , electrospray 
 emitters are not monolithically coupled with lc columns but instead 
 are connected via capillary tubing with proper fittings , which results 
 in dead volumes and postcolumn losses . in addition , the micrometer - size 
 nanospray emitters are easily clogged by plasma proteins because they 
 are denatured under high esi voltage and high organic solvents when 
 eluted from the c4 column . this contributes to the low robustness 
 and lack of reproducibility for nanospray ms and renders it unsuitable 
 for clinical applications . indeed , we observed significant clogging 
 of capillary emitters ( e.g. , picotip ) interfaced to a commercial capillary 
 c4 column during our initial method developments . in contrast , our 
 microfabricated emitters are monolithically interfaced with the on - chip 
 and online c4 column on the silicon - based mea chip . this significantly 
 reduces the clogging and minimizes the dead volume , thereby increasing 
 the sensitivity and robustness while maintaining the specificity and 
 accuracy of our nanoflow lc  ms assay . our mea chip has sustained 
 the same level of performance after over 100 consecutive lc ms 
 runs of crude whole blood samples with essentially no sample cleanup . 
 the 1-h lc ms run was used in this work , but future optimization 
 of the lc method and chip parameters , such as implementation of staggered 
 parallel separation to increase the ms duty cycle , could significantly shorten the total run time down to 
 minutes for faster analysis , to be on par with the turnaround time 
 of immunoassays . implementation of multiplex and multifunction on - chip 
 columns ( e.g. , 24-plex ) on our mea chip will further increase the 
 throughput of our assay for parallel analysis of a large number of 
 samples . our assay could provide new opportunities in understanding 
 the 
 biology and improving the long - term management of diabetes . diabetes 
 ( including type 1 , type 2 , and gestational ) is a very complex and 
 heterogeneous disease . the mechanisms underlying each subtypes of 
 diabetes and how the oxidative stress induces both microvascular and 
 cardiovascular complications of diabetes remain elusive . our assay directly monitors the oxidative stress 
 using one key plasma marker , hsa - cys , and possibly cardiovascular 
 risks of diabetes using three potential markers including hba - sno , 
 gapoa - i , and apoa - i meto , concurrently with the corresponding status 
 of longitudinal blood glycemia ( glucose , hba1c , ga , and gapoa - i ) . in fact , heart attacks account for majority of the deaths in 
 patients with diabetes . both glycation and oxidation of apoa - i may 
 affect the functions of hdl ( a key player in cvd ) . therefore , 
 our assay may facilitate longitudinal investigations of the initiation , 
 progression , and consequences of diabetes for each individual and 
 thus provide new insights at the molecular ( proteins ) , cellular ( rbcs ) , 
 and tissue ( blood ) levels . this in turn might help dissect the mechanisms 
 underlying diabetes and provide better disease management . the new 
 fda guidance to industry for the development of new anti - diabetes 
 therapies mandates that their cardiovascular risks be evaluated concurrently 
 for drug safety , in addition to demonstrating their efficacy of lowering 
 and maintaining blood glucose levels . our method simultaneously measures 
 multiple biomarkers for blood glycemia , oxidative stress , and cardiovascular 
 risks of individuals , using a single lc  ms run starting from 
 a single drop of blood , and therefore may be used in conjunction with 
 cardiac markers such as herg ( human potassium ion channel ) in clinical 
 trials to facilitate new drug developments . if further validated , 
 our assay could be utilized for routine monitoring of patients , for 
 example , in response to different treatment regimens , for personalized 
 management and treatment of diabetes . in summary , we have demonstrated 
 a rapid , sensitive , and specific 
 top - down - proteomics - centric clinical assay for monitoring multiclass 
 biomarkers of diabetes starting from a drop of blood ( 5 l ) 
 using our mea chip platform . if combined with the microsampling of 
 blood ( e.g. , using calibrated capillary tubes ) and validated with 
 a larger patient population in conjunction with prospective clinical 
 studies to determine the limit of detection ( lod ) , inter- and intrapatient 
 variations , within- and between - day reproducibility , and comparability 
 with the existing commercial platforms , our assay may contribute to 
 the long - term management of diabetes and promote the clinical applications 
 of top - down proteomics in theranostics of other diseases , for example , 
 cancer and neurodegenerative diseases .
the 
 most common markers for monitoring patients with diabetes are 
 glucose and hba1c , but additional markers such as glycated human serum 
 albumin ( hsa ) have been identified that could address the glycation 
 gap and bridge the time scales of glycemia between transient and 23 
 months . however , there is currently no technical platform that could 
 measure these markers concurrently in a cost - effective manner . 
 we 
 have developed a new assay that is able to measure glucose , hba1c , 
 glycated hsa , and glycated apolipoprotein a - i ( apoa - i ) for monitoring 
 of individual blood glycemia , as well as cysteinylated hsa , s - nitrosylated 
 hba , and methionine - oxidized apoa - i for gauging oxidative stress and 
 cardiovascular risks , all in 5 l of blood . 
 the assay utilizes 
 our proprietary multinozzle emitter array chip technology to enable 
 the analysis of small volumes of blood , without complex sample preparation 
 prior to the online and on - chip liquid chromatography 
 nanoelectrospray 
 ionization mass spectrometry . 
 importantly , the assay employs top - down 
 proteomics for more accurate quantitation of protein levels and for 
 identification of post - translational modifications . 
 further , the assay 
 provides multimarker , multitime - scale , and multicompartment monitoring 
 of blood glycemia . 
 our assay readily segregates healthy controls from 
 type 2 diabetes patients and may have the potential to enable better 
 long - term monitoring and disease management of diabetes .