MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma

A highly-multiplexed MRM-based assay for determination of cardiovascular disease (CVD) status and disease classification has been developed for clinical research. A high-flow system using ultra-high performance LC and an Agilent 6490 triple quadrupole mass spectrometer, equipped with an ion funnel, provided ease of use and increased the robustness of the assay. The assay uses 135 stable isotope-labeled peptide standards for the quantitation of 67 putative biomarkers of CVD in tryptic digests of whole plasma in a 30-min assay. Eighty-five analyses of the same sample showed no loss of sensitivity (<20% CV for 134/135 peptides) and no loss of retention time accuracy (<0.5% CV for all peptides). The maximum linear dynamic range of the MRM assays ranged from 10(3) -10(5) for 106 of the assays. Excellent linear responses (r >0.98) were obtained for 117 of the 135 peptide targets with attomole level limits of quantitation (<20% CV and accuracy 80-120%) for 81 of the 135 peptides. The assay presented in this study is easy to use, robust, sensitive, and has high-throughput capabilities through short analysis time and complete automated sample preparation. It is therefore well suited for CVD biomarker validation and discovery in plasma.     

An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue

The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics.     

Targeted quantitation of CVD-linked plasma proteins for biomarker verification and validation

Despite significant advances in treatment, cardiovascular disease (CVD) remains one of the leading causes of morbidity and mortality in developed and developing countries. Judicious monitoring of common risk factors has been unable to control this global epidemic, necessitating novel biomarkers for improved screening and earlier disease detection and management. Although numerous plasma proteins have been associated with CVD, only a few of these potential biomarkers have been validated for clinical use. Here we review the quantitative proteomic methods used to verify and validate new biomarker candidates in human plasma. These methods center on a bottom-up approach involving multiple or selected reaction monitoring, for targeted detection, with stable isotope-labeled standards, for peptide normalization. Also included are a discussion of future strategies for improved CVD protein biomarker verification and validation, recommendations for method translation to the clinic, and future projections for protein biomarker research.     

Analytical platform for verification and quantitation of target peptides in human serum: application to cathelicidin

A selective and sensitive, fully automated platform for verification and quantitative determination of target peptides in biofluids is proposed and then validated by development of a method for analysis of cathelicidin in human serum. The method is based on the on-line coupling of solid-phase extraction (SPE) and tandem mass spectrometry with direct infusion. Mass spectrometry analysis was carried out by multiple reaction monitoring using three transitions (one for quantitative analysis and two for qualitative analysis), all them confirmed by in silico fragmentation of the target peptide. Samples were prepared in the SPE workstation on a polymeric divinylbenzene resin by preconcentration, deproteinization, and cleanup, removing salts and interferences after direct injection of human serum. The analytical process required 12 min. The limits of detection and quantitation were 2.5 and 8.25 μg/L, respectively (0.20 and 0.66 pg on column). Repeatability and within-laboratory reproducibility were 2.4% and 2.7%, respectively. A dual-cartridge configuration was used to test recovery of cathelicidin in serum, resulting in 80%. Because quantitative retention in the cartridge was assessed, determination of cathelicidin was validated without using synthetic peptides labeled with stable isotopes. The hyphenated system allows full automation, thereby improving reproducibility and accuracy, as demanded by clinical analysis.     

Multiplexed MRM‐based quantitation of candidate cancer biomarker proteins in undepleted and non‐enriched human plasma

An emerging approach for multiplexed targeted proteomics involves bottom‐up LC‐MRM‐MS, with stable isotope‐labeled internal standard peptides, to accurately quantitate panels of putative disease biomarkers in biofluids. In this paper, we used this approach to quantitate 27 candidate cancer‐biomarker proteins in human plasma that had not been treated by immunoaffinity depletion or enrichment techniques. These proteins have been reported as biomarkers for a variety of human cancers, from laryngeal to ovarian, with breast cancer having the highest correlation. We implemented measures to minimize the analytical variability, improve the quantitative accuracy, and increase the feasibility and applicability of this MRM‐based method. We have demonstrated excellent retention time reproducibility (median interday CV: 0.08%) and signal stability (median interday CV: 4.5% for the analytical platform and 6.1% for the bottom‐up workflow) for the 27 biomarker proteins (represented by 57 interference‐free peptides). The linear dynamic range for the MRM assays spanned four orders‐of‐magnitude, with 25 assays covering a 10³–10⁴ range in protein concentration. The lowest abundance quantifiable protein in our biomarker panel was insulin‐like growth factor 1 (calculated concentration: 127 ng/mL). Overall, the analytical performance of this assay demonstrates high robustness and sensitivity, and provides the necessary throughput and multiplexing capabilities required to verify and validate cancer‐associated protein biomarker panels in human plasma, prior to clinical use.     

Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate

Selected or multiple reaction monitoring is a targeted mass spectrometry method (S/MRM-MS), in which many peptides are simultaneously and consistently analyzed during a single liquid chromatography-mass spectrometry (LC-S/MRM-MS) measurement. These capabilities make S/MRM-MS an attractive method to monitor a consistent set of proteins over various experimental conditions. To increase throughput for S/MRM-MS it is advantageous to use scheduled methods and unfractionated protein extracts. Here, we established the practically measurable dynamic range of proteins reliably detectable and quantifiable in an unfractionated protein extract from a human cell line using LC-S/MRM-MS. Initially, we analyzed S/MRM transition peak groups in terms of interfering signals and compared S/MRM transition peak groups to MS1-triggered MS2 spectra using dot-product analysis. Finally, using unfractionated protein extract from human cell lysate, we quantified the upper boundary of copies per cell to be 35 million copies per cell, while 7500 copies per cell represents a lower boundary using a single 35 min linear gradient LC-S/MRM-MS measurement on a current, standard commercial instrument.     

Robust protein quantitation in chromatographic fractions using MALDI-MS of tryptic peptides

A method is introduced to evaluate protein concentrations using the height sum of all MALDI-MS peaks that unambiguously match theoretic tryptic peptide masses of the protein sought after. The method uses native chromatographic protein fractionation prior to digestion but does not require any depletion, labeling, derivatization, or preparation of a compound similar to the analyte. All peak heights of tryptic peptides are normalized with the peak height of a unique standard peptide added to the MALDI-MS samples. The sum of normalized peak heights, S(n), or the normalized mean peak height, M(n), reflects the concentration of the respective protein. For fractions containing various proteins, S(n) and M(n) can be used to compare concentrations of a protein between different fractions. For fractions with one predominating protein, they can be used to estimate concentration ratios between fractions, or to quantify the fractional protein concentration after calibration with pure protein solutions. Initial native fractionation retains the possibility to apply all conventional analytic procedures. Moreover, it renders the method relatively robust to MS mass accuracy. The method was validated with albumin, transferrin, alpha1-antitrypsin, and immunoglobulin G within highly complex chromatographic fractions of pathological and normal sera, which contained the respective intact native protein in dominating as well as minor concentrations. The correlation found between S(n) and the protein concentration as determined with ELISA showed that the method can be applied to select markers for distinguishing between normal and pathological serum samples.     

Advances in proteomic prostate cancer biomarker discovery

Prostate cancer is the most common non-cutaneous cancer in men in the United States. For reasons largely unknown, the incidence of prostate cancer has increased in the last two decades, in spite or perhaps because of a concomitant increase in serum prostate-specific antigen (PSA) screening. While PSA is acknowledged not to be an ideal biomarker for prostate cancer detection, it is however widely used by physicians due to lack of an alternative. Thus, the identification of a biomarker(s) that can complement or replace PSA represents a major goal for prostate cancer research. Screening complex biological specimens such as blood, urine, and tissue to identify protein biomarkers has become increasingly popular over the last decade thanks to advances in proteomic discovery methods. The completion of human genome sequence together with new development in mass spectrometry instrumentation and bioinformatics has been a major driving force in biomarker discovery research. Here we review the current state of proteomic applications as applied to various sample sources including blood, urine, tissue, and “secretome” for the purpose of prostate cancer biomarker discovery. Additionally, we review recent developments in validation of putative markers, efforts at systems biology approach, and current challenges of proteomics in biomarker discovery.     

Advances in high-resolution quantitative proteomics: implications for clinical applications

The advances in high-resolution mass spectrometry instrumentation, capable of accurate mass measurement and fast acquisition, have enabled new approaches for targeted quantitative proteomics. More specifically, analyses performed on quadrupole-orbitrap mass spectrometers operated in parallel reaction monitoring (PRM) mode leverage the intrinsic high resolving power and trapping capabilities. The PRM technique offers unmatched degrees of selectivity and analytical sensitivity, typically required to analyze peptides in complex samples, such as those encountered in biomedical research or clinical studies. The features of PRM have provoked a paradigm change in targeted experiments, by decoupling acquisition and data processing. It has resulted in a new analytical workflow comprising distinct methods for each step, thus enabling much larger flexibility. The PRM technique was further enhanced by a new data acquisition scheme, allowing dynamic parameter settings. The potential of the technique may radically impact future quantitative proteomics studies.     

Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins

Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents.     

Fluoroprofile, a fluorescence-based assay for rapid and sensitive quantitation of proteins in solution

The development of a sensitive fluorescence-based assay for the quantitative determination of protein concentration is described. The assay is based on the natural product epicocconone, which produces a large increase in fluorescence quantum yield upon binding to detergent-coated proteins in solution. There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 microg/mL for most proteins. There is little protein-to-protein variation except for iron-containing proteins and the assay can be used so that it is tolerant of chemicals commonly used in 2-D sample buffers. The assay is more sensitive than standard absorption assays such as the Bradford and Lowry assays, and has a greater dynamic range and sensitivity than other fluorescent assays.     

Current progress in protein profiling of complex samples

Accurate cancer biomarkers are needed for early detection, disease classification, prediction of therapeutic response and monitoring treatment. While there appears to be no shortage of candidate biomarker proteins, a major bottleneck in the biomarker pipeline continues to be their verification by enzyme linked immunosorbent assays. Multiple reaction monitoring (MRM), also known as selected reaction monitoring, is a targeted mass spectrometry approach to protein quantitation and is emerging to bridge the gap between biomarker discovery and clinical validation. Highly multiplexed MRM assays are readily configured and enable simultaneous verification of large numbers of candidates facilitating the development of biomarker panels which can increase specificity. This review focuses on recent applications of MRM to the analysis of plasma and serum from cancer patients for biomarker verification. The current status of this approach is discussed along with future directions for targeted mass spectrometry in clinical biomarker validation.     

The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

Mass spectrometry (MS) -based proteomics has become an indispensable tool with broad applications in systems biology and biomedical research. With recent advances in liquid chromatography (LC) and MS instrumentation, LC–MS is making increasingly significant contributions to clinical applications, especially in the area of cancer biomarker discovery and verification. To overcome challenges associated with analyses of clinical samples (for example, a wide dynamic range of protein concentrations in bodily fluids and the need to perform high throughput and accurate quantification of candidate biomarker proteins), significant efforts have been devoted to improve the overall performance of LC–MS-based clinical proteomics platforms. Reviewed here are the recent advances in LC–MS and its applications in cancer biomarker discovery and quantification, along with the potentials, limitations and future perspectives.     

An integrated quantification method to increase the precision,robustness, and resolution of protein measurement in human plasma samples

Background

Current quantification methods for mass spectrometry (MS)-based proteomics either do not provide sufficient control of variability or are difficult to implement for routine clinical testing.

Results

We present here an integrated quantification (InteQuan) method that better controls pre-analytical and analytical variability than the popular quantification method using stable isotope-labeled standard peptides (SISQuan). We quantified 16 lung cancer biomarker candidates in human plasma samples in three assessment studies, using immunoaffinity depletion coupled with multiple reaction monitoring (MRM) MS. InteQuan outperformed SISQuan in precision in all three studies and tolerated a two-fold difference in sample loading. The three studies lasted over six months and encountered major changes in experimental settings. Nevertheless, plasma proteins in low ng/ml to low μg/ml concentrations were measured with a median technical coefficient of variation (CV) of 11.9% using InteQuan. The corresponding median CV using SISQuan was 15.3% after linear fitting. Furthermore, InteQuan surpassed SISQuan in measuring biological difference among clinical samples and in distinguishing benign versus cancer plasma samples.

Conclusions

We demonstrated that InteQuan is a simple yet robust quantification method for MS-based quantitative proteomics, especially for applications in biomarker research and in routine clinical testing.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-12-3) contains supplementary material, which is available to authorized users.     

An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum

An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap-release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3 nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay.     

Is a gene‐centric human proteome project the best way for proteomics to serve biology?

With the recent developments in proteomic technologies, a complete human proteome project (HPP) appears feasible for the first time. However, there is still debate as to how it should be designed and what it should encompass. In “proteomics speak”, the debate revolves around the central question as to whether a gene‐centric or a protein‐centric proteomics approach is the most appropriate way forward. In this paper, we try to shed light on what these definitions mean, how large‐scale proteomics such as a HPP can insert into the larger omics chorus, and what we can reasonably expect from a HPP in the way it has been proposed so far.     

Post-translational quantitation by SRM/MRM: applications in cardiology

Introduction: Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization, and interaction with other molecules. PTMs apply a dynamic control of proteins in both physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution mass spectrometry (MS)/MS analysis provide attractive results on PTM characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples.

Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported, and a specific section is focused on PTM detection in proteins that are involved in the cardiovascular system and heart diseases.

Expert commentary: PTM characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded our knowledge in the last few years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTM complexity. Promising studies confirm the great potential of SRM/MRM to study PTMs in the cardiovascular field, and PTMomics could be very useful in the clinical perspective.     

Plasma protein thiolation index (PTI) as a biomarker of thiol-specific oxidative stress in haemodialyzed patients

The role of oxidative stress in patients with end stage renal disease (ESRD), which occurs at significantly higher levels than in the general population, is often underestimated in clinical practice. Emerging evidence highlights the strong correlation of oxidative stress with chronic inflammation and cardiovascular disease, which are highly prevalent in most patients on maintenance haemodialysis (HD) and are a major risk factor for mortality in this population. In this study, total plasma thiols and plasma S-thiolated proteins were measured in patients with ESRD, before and after a regular HD session, and compared to age-matched healthy subjects. We found a significant decrease in the level of total plasma thiols and, conversely, a significant increase in the level of S-thiolated proteins in these patients. In most patients, post-HD plasma level of total thiols did not differ from the one in healthy subjects, whereas plasma level of S-thiolated proteins was lower in HD patients than in age-matched healthy controls. This suggests that a single HD session restores plasma thiol redox status and re-establishes the antioxidant capacity of plasma thiols. Additionally, we determined protein thiolation index (PTI), i.e., the molar ratio between the sum of all low molecular mass thiols bound to S-thiolated plasma proteins and protein free cysteinyl residues. Patients with ESRD had a significantly higher PTI compared to age-matched healthy subjects and HD was associated with a decrease in PTI to normal, or lower than normal, levels. Although this study is limited in size, our results suggest that PTI is a useful indicator of thiol-specific oxidative stress in patients with ESRD on maintenance HD. This study also emphasizes that PTI determination is a cheap and simple tool suitable for large-scale clinical studies that could be used for routine screening of thiol-specific oxidative stress.     

Prospective applications of ultrahigh resolution proteomics in clinical mass spectrometry

Introduction: Advances in mass spectrometry (MS)-based proteomic strategies have resulted in robust protein biomarker discovery studies often performed on high resolution accurate mass (HRAM) platforms. For successful translation of promising protein biomarkers into useful clinical tests, trans-sector networks and collaboration among stakeholders involved in the biomarker pipeline are urgently needed.

Areas covered: In this perspective, literature- and empirical evidence is combined with author’s opinions to discuss the progress of ultrahigh resolution MS and provide insight in its potential for validation and development of clinical tests.

Expert commentary: Thus far two ‘low resolution’ MS strategies have been implemented in the clinic: quantification of proteins using triple quadrupole instruments and identification of unknown microorganisms using comparative analysis with spectral libraries on MALDI-TOF instruments. The rise of HRAM technology further boosts the potential of MS-based tests for detection and quantitation of disease-specific biomarkers which meet the analytical performance specifications needed for clinical assays.     

Distribution and properties of protein kinase and protein phosphatase activities in synaptosomal plasma membranes and synaptic junctions

Some characteristics of the protein kinase activity associated with a synaptosomal plasma membrane (synaptic membrane) fraction and a synaptic junction fraction have been compared. Autoradiography of the phosphorylated fractions separated on sodium dodecyl sulfate polyacrylamine gels showed that cyclic AMP stimulates the phosphorylation of five polypeptides in synaptic membranes, whereas no cyclic AMP dependency could be detected in synaptic junctions. Kinetic studies demonstrated that synaptic junctions contain at high K_m and a low K_m protein kinase activity while only the high K_m activity could be detected in synaptic membranes. The intrinsic ATPase activity of synaptic membranes was shown to strongly interfere with measurements of protein kinase activity. Cyclic AMP binding experiments revealed a 2.6-fold enrichment of cyclic AMP binding capacity in synaptic junctions as compared to synaptic membranes. Protein phosphatase activity was not detected in synaptic junctions but was associated with synaptic membranes, where cyclic AMP was shown to either stimulate or inhibit the dephosphorylation of different polypeptides.