Plasma Peptidome Research Articles

Comparison of Urine and Plasma Peptidome Indicates Selectivity in Renal Peptide Handling

Urine is considered to be produced predominantly as a result of plasma filtration in the kidney. However, the origin of the native peptides present in urine has never been investigated in detail. Therefore, the authors aimed to obtain a first insight into the origin of urinary peptides based on a side-by-side comprehensive analysis of the plasma and urine peptidome. Twenty-two matched urine and plasma samples are analyzed for their peptidome using capillary electrophoresis coupled to mass spectrometry (CE-MS; for relative quantification) and CE or LC coupled to tandem mass spectrometry (CE- or LC-MS/MS; for peptide identification). The overlap and association of abundance of the different peptides present in these two body fluids are evaluated. The authors are able to identify 561 plasma and 1461 urinary endogenous peptides. Only 90 peptides are detectable in both urine and plasma. No significant correlation is found when comparing the abundance of these common peptides, with the exception of collagen fragments. This observation is also supported when comparing published peptidome data from both plasma and urine. Most of the plasma peptides are not detectable in urine, possibly due to tubular reabsorption. The majority of urinary peptides may in fact originate in the kidney. The notable exception is collagen fragments, which indicates potential selective exclusion of these peptides from tubular reabsorption. Experimental verification of this hypothesis is warranted.

Multiplexed Temporal Quantification of the Exercise-regulated Plasma Peptidome

Exercise is extremely beneficial to whole body health reducing the risk of a number of chronic human diseases. Some of these physiological benefits appear to be mediated via the secretion of peptide/protein hormones into the blood stream. The plasma peptidome contains the entire complement of low molecular weight endogenous peptides derived from secretion, protease activity and PTMs, and is a rich source of hormones. In the current study we have quantified the effects of intense exercise on the plasma peptidome to identify novel exercise regulated secretory factors in humans. We developed an optimized 2D-LC-MS/MS method and used multiple fragmentation methods including HCD and EThcD to analyze endogenous peptides. This resulted in quantification of 5,548 unique peptides during a time course of exercise and recovery. The plasma peptidome underwent dynamic and large changes during exercise on a time-scale of minutes with many rapidly reversible following exercise cessation. Among acutely regulated peptides, many were known hormones including insulin, glucagon, ghrelin, bradykinin, cholecystokinin and secretogranins validating the method. Prediction of bioactive peptides regulated with exercise identified C-terminal peptides from Transgelins, which were increased in plasma during exercise. In vitro experiments using synthetic peptides identified a role for transgelin peptides on the regulation of cell-cycle, extracellular matrix remodeling and cell migration. We investigated the effects of exercise on the regulation of PTMs and proteolytic processing by building a site-specific network of protease/substrate activity. Collectively, our deep peptidomic analysis of plasma revealed that exercise rapidly modulates the circulation of hundreds of bioactive peptides through a network of proteases and PTMs. These findings illustrate that peptidomics is an ideal method for quantifying changes in circulating factors on a global scale in response to physiological perturbations such as exercise. This will likely be a key method for pinpointing exercise regulated factors that generate health benefits.

Chronological Profiling of Plasma Native Peptides after Hepatectomy in Pigs: Toward the Discovery of Human Biomarkers for Liver Regeneration.

Liver regeneration after partial hepatectomy (PHx) is a time-dependent process, which is tightly regulated by multiple signaling cascades. Failure of this complex process leads to posthepatectomy liver failure (PHLF), which is associated with a high rate of mortality. Thus, it is extremely important to establish a useful biomarker of liver regeneration to help prevent PHLF. Here, we hypothesized that alterations in the plasma peptide profile may predict liver regeneration following PHx and hence we set up a diagnostic platform for monitoring posthepatectomy outcome. We chronologically analyzed plasma peptidomic profiles of 5 partially hepatectomized microminipigs using the ClinProtTM system, which consists of magnetic beads and MALDI-TOF/TOF MS. We identified endogenous circulating peptides specific to each phase of the postoperative course after PHx in pigs. Notably, peptide fragments of histones were detected immediately after PHx; the presence of these fragments may trigger liver regeneration in the very acute phase after PHx. An N-terminal fragment of hemoglobin subunit α (3627 m/z) was detected as an acute-phase-specific peptide. In the recovery phase, the short N-terminal fragments of albumin (3028, 3042 m/z) were decreased, whereas the long N-terminal fragment of the protein (8926 m/z) was increased. To further validate and extract phase-specific biomarkers using plasma peptidome after PHx, plasma specimens of 4 patients who underwent PHx were analyzed using the same method as we applied to pigs. It revealed that there was also phase-specificity in peptide profiles, one of which was represented by a fragment of complement C4b (2378 m/z). The strategy described herein is highly efficient for the identification and characterization of peptide biomarkers of liver regeneration in a swine PHx model. This strategy is feasible for application to human biomarker studies and will yield clues for understanding liver regeneration in human clinical trials.

Characterization of the Low-Molecular-Weight Human Plasma Peptidome.

The human plasma proteome represents an important secreted sub-proteome. Proteomic analysis of blood plasma with mass spectrometry is a challenging task. The high complexity and wide dynamic range of proteins as well as the presence of several proteins at very high concentrations complicate the profiling of the human plasma proteome. The peptidome (or low-molecular-weight fraction, LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based markers of disease. Peptides are generated by active synthesis and proteolytic processing, often yielding proteolytic fragments that mediate a variety of physiological and pathological functions. As such, degradomic studies, investigating cleavage products via peptidomics and top-down proteomicsin particular, have warranted significant research interest. However, due to their molecular weight, abundance, and solubility, issues with identifying specific cleavage sites and coverage of peptide fragments remain challenging. Peptidomics is currently focused toward comprehensively studying peptides cleaved from precursor proteins by endogenous proteases. This protocol outlines a standardized rapid and reproducible procedure for peptidomic profiling of human plasma using centrifugal ultrafiltration and mass spectrometry. Ultrafiltration is a convective process that uses anisotropic semipermeable membranes to separate macromolecular species on the basis of size. We have optimized centrifugal ultrafiltration (cellulose triacetate membrane) for plasma fractionation with respect to buffer and solvent composition, centrifugal force, duration, and temperature to facilitate recovery >95% and enrichment of the human plasma peptidome. This method serves as a comprehensive and facile process to enrich and identify a key, underrepresented sub-proteome of human blood plasma.

Set up of a protocol for rat plasma peptidomics in hemorrhagic shock model in presence of heparin

A preliminary mass spectrometry based shotgun protocol was set up to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats with the aim of verifying the possible role of uncontrolled proteolytic activity in circulatory shock. Since the hemorrhagic shock model requires heparin as anticoagulant, a preliminary experiment using plasma sample obtained in the presence/absence of heparin from healthy rats was performed to determine whether its presence is fully compatible with the peptidomic protocol proposed. The entire protocol was tested in a pilot experiment to compare the peptidome of healthy or heparin-anticoagulated rats subjected to hemorrhagic shock.

Alterations of the Plasma Peptidome Profiling in Colorectal Cancer Progression.

Early detection of colorectal cancer (CRC) remains a challenge. It has been highlighted that the pathological alterations within an organ and tissues might be reflected in serum or plasma proteomic/peptidic patterns. The aim of the study was to follow the changes in the plasma peptides associated to colorectal cancer progression by mass spectrometry. This study included 27 adenoma, 67 CRC (n = 33 I-II stage and n = 34 III-IV stage), 23 liver metastasis from CRC patients and 34 subjects disease-free as controls. For plasma peptides analysis, samples purification was performed on the Nanoporous Silica Chips technology followed by matrix-assisted laser desorption/ionisation-time of flight analysis. Since the high complexity of the obtained dataset, multivariate statistical analysis, and discriminant pattern recognition were performed for study groups classification. Forty-four of 88 ionic species were successfully identified as fragments of peptides and proteins physiologically circulating in the blood and belonging to immune and coagulation systems and inflammatory mediators. Many peptides clustered into sets of overlapping sequences with ladder-like truncation clearly associated to proteolytic processes of both endo- and exoproteases activity. Comparing to controls, a different median ion intensity of the group-type fragments distribution was observed. Moreover, the degradation pattern obtained by proteolytic cleavage was different into study groups. This pattern was specific and characteristic of each group: controls, colon tumour disease (including adenoma and CRC), and liver metastasis, revealing a role as biomarker in early diagnosis and prognosis. Our findings highlighted peculiar changes in protease activity characteristic of CRC progression from pre-cancer lesion to metastatic disease. J. Cell. Physiol. 231: 915-925, 2016. © 2015 Wiley Periodicals, Inc.

Mass spectrometry-based serum and plasma peptidome profiling for prediction of treatment outcome in patients with solid malignancies.

Treatment selection tools are needed to enhance the efficacy of targeted treatment in patients with solid malignancies. Providing a readout of aberrant signaling pathways and proteolytic events, mass spectrometry-based (MS-based) peptidomics enables identification of predictive biomarkers, whereas the serum or plasma peptidome may provide easily accessible signatures associated with response to treatment. In this systematic review, we evaluate MS-based peptide profiling in blood for prompt clinical implementation. PubMed and Embase were searched for studies using a syntax based on the following hierarchy: (a) blood-based matrix-assisted or surface-enhanced laser desorption/ionization time-of-flight MS peptide profiling (b) in patients with solid malignancies (c) prior to initiation of any treatment modality, (d) with availability of outcome data. Thirty-eight studies were eligible for review; the majority were performed in patients with non-small cell lung cancer (NSCLC). Median classification prediction accuracy was 80% (range: 66%-93%) in 11 models from 14 studies reporting an MS-based classification model. A pooled analysis of 9 NSCLC studies revealed clinically significant median progression-free survival in patients classified as "poor outcome" and "good outcome" of 2.0 ± 1.06 months and 4.6 ± 1.60 months, respectively; median overall survival was also clinically significant at 4.01 ± 1.60 months and 10.52 ± 3.49 months, respectively. Pretreatment MS-based serum and plasma peptidomics have shown promising results for prediction of treatment outcome in patients with solid tumors. Limited sample sizes and absence of signature validation in many studies have prohibited clinical implementation thus far. Our pooled analysis and recent results from the PROSE study indicate that this profiling approach enables treatment selection, but additional prospective studies are warranted.

Semi-quantitative analysis of changes in the plasma peptidome of Manduca sexta larvae and their correlation with the transcriptome variations upon immune challenge

The tobacco hornworm, Manduca sexta, has been used as a biochemical model for studying insect physiological processes. While the transcriptomes of its fat body, hemocytes, midgut, and antennae have been examined in several studies, limited information is available for proteins in tissues, cells, or body fluids of this insect. In keeping pace with the M. sexta genome project, we launched a pilot study to identify differences in the peptidome of cell-free hemolymph samples from larvae injected with buffer or a mixture of bacteria. At 24 h after injection, plasma was collected and treated with 50% acetonitrile to precipitate large proteins. The supernatants, containing peptides (<25 kDa) and other stable proteins (>25 kDa), were digested with trypsin and analyzed by nano-liquid chromatography and nano-electrospray tandem mass spectrometry (nanoLC-MS/MS) on an LTQ Orbitrap XL mass spectrometer. Known M. sexta cDNA sequences and gene transcripts from the draft genome were translated in silico to generate a database of polypeptides (i.e. peptides and proteins) in this species. By searching the database, we identified 268 hemolymph polypeptides, 50 of which showed 1.67–200 fold abundance increases after the immune challenge, as judged by significant changes in normalized spectral counts between the control and induced plasma. These included a total of 33 antimicrobial peptides (attacins, cecropins, defensins, diapausins, gallerimycin, gloverin, lebocins, lysozymes), pattern recognition receptors, and proteinase inhibitors. Although there was no strong parallel (correlation coefficients: −0.13, 0.11, 0.39 and 0.62) between plasma peptide levels and their transcript levels in control or induced hemocytes or fat body, we observed the mRNA level changes in hemocytes and fat body concurred with their peptide level changes with correlation coefficients of 0.67 and 0.76, respectively. These data suggest that fat body contributed a significant portion of the plasma polypeptides involved in various aspects of innate immunity after the bacterial injection.

Detection of Cervical Cancer Biomarker Patterns in Blood Plasma and Urine by Differential Scanning Calorimetry and Mass Spectrometry

Improved methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN) and extent of spread of invasive carcinomas of the cervix (IC) are needed. Differential scanning calorimetry (DSC) has recently been shown to detect specific changes in the thermal behavior of blood plasma proteins in several diseases. This methodology is being explored to provide a complementary approach for screening of cervical disease. The present study evaluated the utility of DSC in differentiating between healthy controls, increasing severity of CIN and early and advanced IC. Significant discrimination was apparent relative to the extent of disease with no clear effect of demographic factors such as age, ethnicity, smoking status and parity. Of most clinical relevance, there was strong differentiation of CIN from healthy controls and IC, and amongst patients with IC between FIGO Stage I and advanced cancer. The observed disease-specific changes in DSC profiles (thermograms) were hypothesized to reflect differential expression of disease biomarkers that subsequently bound to and affected the thermal behavior of the most abundant plasma proteins. The effect of interacting biomarkers can be inferred from the modulation of thermograms but cannot be directly identified by DSC. To investigate the nature of the proposed interactions, mass spectrometry (MS) analyses were employed. Quantitative assessment of the low molecular weight protein fragments of plasma and urine samples revealed a small list of peptides whose abundance was correlated with the extent of cervical disease, with the most striking plasma peptidome data supporting the interactome theory of peptide portioning to abundant plasma proteins. The combined DSC and MS approach in this study was successful in identifying unique biomarker signatures for cervical cancer and demonstrated the utility of DSC plasma profiles as a complementary diagnostic tool to evaluate cervical cancer health.

Predictive response biomarkers in rectal cancer neoadjuvant treatment

Locally advanced rectal cancer (RC) treatment is a challenge, because RC has a high rate of local recurrence. To date preoperative chemoradiotherapy (pCRT) is widely accepted as standard protocol of care for middle-low RC, but complete tumour response rate ranges from 4 to 44% and 5-year local recurrence rate is 6%. Better understanding of molecular biology and carcinogenesis pathways could be used both for pre-neoplastic lesions and locally recurrence diagnosis, and for tumour response prediction to therapy. Circulating molecules, gene expression and protein signature are promising sources to biomarker discovery. Several studies have evaluated potential predictors of response and recently, cell-free Nucleic Acid levels have been associated to tumour response to neoadjuvant therapies. Alternative method is the serum or plasma proteome and peptidome analysis. It may be ideally suited for its minimal invasiveness and it can be repeated at multiple time points throughout the treatment in contrast to tissue-based methods which still remain the most reliable and specific approach. Many studies have analyzed preoperative rectal tissue prognostic factor, but data are controversial or not confirmed.

Calorimetric analysis of the plasma proteome: Identification of type 1 diabetes patients with early renal function decline

BackgroundMicroalbuminuria (MA) has been questioned as a predictor of progressive renal dysfunction in patients with type 1 diabetes (T1D). Consequently, new clinical end points are needed that identify or predict patients that are at risk for early renal function decline (ERFD). The potential clinical utility of differential scanning calorimetry (DSC) analysis of blood plasma and other biofluids has recently been reported. This method provides an alternate physical basis with which to study disease-associated changes in the bulk plasma proteome. MethodsDSC analysis of blood plasma was applied to identify unique signatures of ERFD in subjects enrolled in the 1st Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a prospective cohort study of T1D patients. Recent data suggests that differences in the plasma peptidome of these patients correlate with longitudinal measures of renal function. Differences in DSC profile (thermogram) features were evaluated between T1D MA individuals exhibiting ERFD (n=15) and matched control subjects (n=14). ResultsThe average control group thermogram resembled a previously defined healthy thermogram. Differences were evident between ERFD and control individuals. Heat capacity values of the main two transitions were found to be significant discriminators of patient status. ConclusionsResults from this pilot study suggest the potential utility of DSC proteome analysis to prognostic indicators of renal disease in T1D. General significanceDSC shows sensitivity to changes in the bulk plasma proteome that correlate with clinical status in T1D providing additional support for the utility of DSC profiling in clinical diagnostics.

Magnetic bead-based proteomic technology to study paricalcitol effect in kidney transplant recipients

Secondary hyperparathyroidism is a common complication in patients with chronic kidney disease and frequently persists after kidney transplantation. Paricalcitol, a selective vitamin D receptor activator, is indicated in the management of this disorder and recent evidences have suggested that this drug has other beneficial effects. Aiming to elucidate these effects, our study included 52 stable kidney transplant recipients randomized 2:1 to treatment with paricalcitol or to no treatment. Bone mineral parameters, kidney function and inflammatory status were assessed at baseline, at 3 and at 12 months. Moreover, a proteomic approach, based on magnetic beads technology coupled to MALDI-TOF mass spectrometry readout, was used to determine changes in patients' plasma peptidome. Patients treated with paricalcitol showed a significant decrease in parathyroid hormone and alkaline phosphatase levels, and an increase of bone mineral density and glomerular filtration rate. The proteomic analysis revealed a decrease in bradykinin after paricalcitol treatment, whereas 2 peptides identified as fragments of the complement factor C4 decreased only in those patients not treated with paricalcitol. These findings suggest that paricalcitol may offer additional benefits due to immunomodulatory effects via the kallikrein-kinin and complement systems.

An Assessment of Peptide Enrichment Methods Employing mTRAQ Quantification Approaches

The human plasma peptidome has potential in biomarker discovery not least because the plasma proteome is a challenging matrix due to its complexity and dynamic range. However, methods to significantly reduce the amount of protein present in plasma while retaining the less abundant peptides present in plasma samples has been a major issue. Here, we present a novel strategy which has been employed to assess the effectiveness of removing interfering proteins while retaining peptides of interest. To monitor peptide retention, a spiked in digested protein, in this case a synthetic QconCAT protein, was employed. This enabled a variety of target analytes (peptides) to be monitored for their retention in liquid phase, providing a broader picture of peptide loss from each method assessed. The incorporation of mTRAQ labeling allowed the presence of each peptide to be monitored, and accurate peptide losses to be determined in a Selected Reaction Monitoring (SRM) assay, thus, enabling an objective semiquantitative conclusion to be drawn regarding the suitability of each method for protein removal and peptide retention. We also assessed a range of methods for retaining nontryptic peptides in a plasma peptidomics workflow. From these data, we determined an optimal workflow for removing intact protein, while retaining peptides for MS-based analyses.

Plasma peptidome profiling of acute hepatitis E patients by MALDI-TOF/TOF

BackgroundHepatitis E is endemic to resource-poor regions, where it manifests as sporadic cases and large waterborne outbreaks. The disease severity ranges from acute self-limited hepatitis with low mortality to fulminant hepatic failure with high mortality. It is believed that the host response plays an important role in determining the progression and outcome of this disease. We profiled the plasma peptidome from hepatitis E patients to discover suitable biomarkers and understand disease pathogenesis.ResultsThe peptidome (< 10 kDa) fraction of plasma was enriched and analyzed by mass spectrometry. A comparative analysis of the peptide pattern of hepatitis E patients versus healthy controls was performed using ClinPro Tools. We generated a peptide profile that could be used for selective identification of hepatitis E cases. We have identified five potential biomarker peaks with m/z values of 9288.6, 7763.6, 4961.5, 1060.572 and 2365.139 that can be used to reliably differentiate between hepatitis E patients and controls with areas under the receiver operating characteristic curve (AUROC) values of 1.00, 0.954, 0.989, 0.960 and 0.829 respectively. A number of proteins involved in innate immunity were identified to be differentially present in the plasma of patients compared to healthy controls.ConclusionsBesides the utility of this approach for biomarker discovery, identification of changes in endogenous peptides in hepatitis E patient plasma has increased our understanding of disease pathogenesis. We have identified peptides in plasma that can reliably distinguish hepatitis E patients from healthy controls. Results from this and an earlier proteomics study are discussed.

Identification of Disulfide Bonds in Protein Proteolytic Degradation Products Using de novo-Protein Unique Sequence Tags Approach

Disulfide bonds are a form of post-translational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags), we unambiguously correlated the spectra to specific database proteins. Examination of the UStags' prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to approximately 10 kDa.

Finding diabetic nephropathy biomarkers in the plasma peptidome by high‐throughput magnetic bead processing and MALDI‐TOF‐MS analysis

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease and improved biomarkers would help identify high-risk individuals. The aim of this study was to discover candidate biomarkers for DN in the plasma peptidome in an in-house cross-sectional cohort (n=122) of type 1 diabetic patients diagnosed with normo-, micro-, and macroalbuminuria. Automated, high-throughput, and reproducible (interassay median CV: 13-14%) plasma peptide profiling protocols involving RPC18 and weak cation exchange magnetic beads on a liquid handling workstation with a MALDI-TOF-MS readout were successfully established. Using these protocols and a combined univariate (Kruskal-Wallis) and multivariate (independent component analysis) statistical analysis approach, ten single peptides and three multi-peptide candidate biomarkers were found. Employment of RPC18 and weak cation exchange magnetic beads proved to be complementary. The proteins found in this study, including C3f and apolipoprotein C-I, represent new candidate biomarkers for DN from the plasma peptidome. The automated procedures and implementation of independent components analysis provide a fast and informative system for analyzing individual patient samples in protein biomarker discovery.

Standardized sample preparation phases for a quantitative measurement of plasma peptidome profiling by MALDI-TOF

A growing body of literature defines MALDI-TOF MS as a technique for studying plasma and serum, thus enabling the detection of proteins, and the generation of reproducible protein profile mass spectra, potentially able to discriminate correctly different biological systems. In this work, the different steps of the pre-analytical phase that may affect the reproducibility of plasma proteome analysis have been carefully considered. The results showed that the method is highly accurate (9.1%) and precise (8.9%) and the calibration curve for the ACTH (18-39), in human plasma, gave a good correlation coefficient (r>0.99 and r(2)>0.98). The limit of detection (LOD) and the limit of quantification (LOQ), relative intensity, were of 0.5 x 10(-)(9)M and 1.0 x 10(-)(9)M respectively. Thus, an assay has been developed for the detection of low-abundant and low molecular weight proteins, from human plasma, aiming at the identification of new potential biomarkers. The method was tested on plasma from patients with a first diagnosis of pelvic mass. Statistical analysis of plasma profile generated a sub-profile of 17 peptides with their relative abundance able to discriminate patients bearing malignant or benign tumors. The sensitivity and specificity were 85.7% and 80.0% respectively.

Analysis of the Plasma Peptidome from Pancreas Cancer Patients Connects a Peptide in Plasma to Overexpression of the Parent Protein in Tumors

Blood circulates through nearly every organ including tumors. Therefore, plasma is a logical source to search for tumor-derived proteins and peptides. The challenge with plasma is that it is a complex bodily fluid composed of high concentrations of normal host proteins that obscure identification of tumor-derived molecules. To simplify plasma, we examined a low molecular weight (LMW) fraction (plasma peptidome) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. In the plasma peptidome of patients with ductal adenocarcinoma of the pancreas (DAP), a prominent peptide was identified from the QSOX1 parent protein. This peptide is stable in whole blood over 24 h and was present in 16 of 23 DAP patients and 4 of 5 patients with intraductal papillary mucinous neoplasm (IPMN). QSOX1 peptides were never identified in the plasma peptidome from 42 normal healthy donors using the same methods. Immunohistochemical staining of DAP tissue sections with anti-QSOX1 antibody shows overexpression of QSOX1 in tumor but not in adjacent stroma or normal ducts. Three of four pancreas tumor cell lines also express QSOX1 protein by Western blot analysis. This is the first report of QSOX1 peptides in plasma from DAP patients and makes the rare connection between a peptide in plasma from cancer patients and overexpression of the parent protein in tumors.

An integrated LC‐ESI‐MS platform for quantitation of serum peptide ladders. Application for colon carcinoma study

Mounting evidence indicates that MS analysis of the human blood peptidome allows to distinguish between cancer and non-cancer samples, giving promise for a new MS-based diagnostic tool. However, several aspects of already published work have been criticized and demand for more methodical approach has been formulated. Motivated by this we undertook a systematic study of the plasma and serum peptidome using an integrated ESI-LC-MS-based platform, equipped with new data analysis tools for relative and absolute peptide quantitation. We used a high resolution LC-ESI-MS to analyze well-separated MS signals corresponding to peptides, and measured the variability of >1000 peptide signal amplitudes across a set of plasma and serum samples from healthy individuals. By spiking serum samples with known amounts of isotopically labeled versions of a selected set of peptides we measured the variability of their absolute concentration in this sample set and demonstrated a strong influence of clotting time on the concentration of these peptides in serum. Finally, we used this new LC-ESI-MS analytical platform for the differential analysis of healthy versus colon cancer serum samples and found that it was possible to distinguish the two groups with 89.8% sensitivity and 94.6% specificity.

Time-Dependent and Sample-to-Sample Variations in Human Plasma Peptidome are Both Minimized Through Use of Protease Inhibitors

In the current study, we have developed a method to measure relative peptide stability over time in different blood collection tubes. Reversed-phase chromatography and liquid chromatography–matrix-assisted laser desorption/ionization were performed on three subjects to facilitate a deeper look into the plasma peptidome. The data further support the importance of protease inhibitors in stabilizing plasma samples. Significantly, we have revealed subject-to-subject variability in the intrinsic damage over time that is possible in standard EDTA plasma, with such variations extensively minimized by protease inhibitors. We conclude that protease inhibitors can simultaneously improve time-dependent and individual-dependent preanalytical variables in human plasma samples.