Clinical Applications Of Proteomics Research Articles

Evaluation of Serum Proteome Sample Preparation Methods to Support Clinical Proteomics Applications.

Serum contains several proteins that are associated with disease-related processes. Mass spectrometry (MS)-based proteomics approaches greatly facilitate serum protein biomarker development. However, the serum proteome complexity presents a technical challenge for the accurate, sensitive, and reproducible quantification of proteins by MS. Thus, efficient sample preparation methods are of critical importance for serum proteome analyses. In this study, we evaluated the technical performance of two serum proteome sample preparation methods using sera from patients with high-grade serous ovarian cancer and patients with benign nongynecological conditions with a goal of providing insight into their compatibility with clinical proteomics workflows. One method entailed the use of immobilized trypsin (SMART Digest Trypsin) with RapiGest SF, an acid-labile surfactant designed to enhance the in-solution enzymatic digestion of proteins. The other method incorporated a commercially available sample preparation kit, iST-BCT, which contains standardized reagents. Significantly higher protein sequence coverage, albeit with lower digestion efficiency, was obtained with the immobilized trypsin + RapiGest SF workflow, whereas the iST-BCT workflow was quicker and had marginally better reproducibility. Protein relative abundance analysis revealed that the serum proteomes clustered primarily by the sample processing workflow and secondarily by disease state. We conducted a time course study to determine whether differences in the relative abundance of diagnostic high-grade serous ovarian cancer serum protein biomarker candidates were biased according to the duration of enzymatic digestion. Our results highlight the importance of optimizing enzymatic digestion kinetics according to the peptide targets of interest while considering the sensitivity of the downstream analytical method utilized in clinical proteomics workflows designed to measure biomarkers.

The journey towards clinical adoption of MALDI-MS-based imaging proteomics: from current challenges to future expectations.

Among the spatial omics techniques available, mass spectrometry imaging (MSI) represents one of the most promising owing to its capability to map the distribution of hundreds of peptides and proteins, as well as other classes of biomolecules, within a complex sample background in a multiplexed and relatively high-throughput manner. In particular, matrix-assisted laser desorption/ionisation (MALDI-MSI) has come to the fore and established itself as the most widely used technique in clinical research. However, the march of this technique towards clinical utility has been hindered by issues related to method reproducibility, appropriate biocomputational tools, and data storage. Notwithstanding these challenges, significant progress has been achieved in recent years regarding multiple facets of the technology and has rendered it more suitable for a possible clinical role. As such, there is now more robust and extensive evidence to suggest that the technology has the potential to support clinical decision-making processes under appropriate circumstances. In this review, we will discuss some of the recent developments that have facilitated this progress and outline some of the more promising clinical proteomics applications which have been developed with a clear goal towards implementation in mind.

NHS-Ester Tandem Labeling in One Pot Enables 48-Plex Quantitative Proteomics.

Stable-isotope labeling strategies are extensively used for multiplex quantitative proteomics. Hybrid-isotope labeling strategies that combine the use of isotopic mass difference labeling and isobaric tags can greatly increase sample multiplexity. In this work, we present a novel hybrid-isotope labeling approach that we termed NHS-ester tandem labeling in one pot (NETLOP). We first optimized 16-plex isobaric TMTpro labeling of lysine residues followed by 2-plex or 3-plex isotopic mTRAQ labeling of peptide N-termini, both of which with commercially available NHS-ester reactive reagents. We then demonstrated the utility of the NETLOP approach by labeling HeLa cell samples and performing proof-of-principle quantitative 32-plex and 48-plex proteomic analyses, each in a single LC-MS/MS experiment. Compared to current hybrid-isotope labeling methods, our NETLOP approach requires no sample cleanup between different labeling steps to minimize sample loss, induces no retention time shifts that compromise quantification accuracy, can be adapted to other NHS-ester isotopic labeling reagents to further increase multiplexity, and is compatible with samples from any origin in a wide array of biological and clinical proteomics applications.

A rigorous evaluation of optimal peptide targets for MS-based clinical diagnostics of Coronavirus Disease 2019 (COVID-19)

BackgroundThe Coronavirus Disease 2019 (COVID-19) global pandemic has had a profound, lasting impact on the world's population. A key aspect to providing care for those with COVID-19 and checking its further spread is early and accurate diagnosis of infection, which has been generally done via methods for amplifying and detecting viral RNA molecules. Detection and quantitation of peptides using targeted mass spectrometry-based strategies has been proposed as an alternative diagnostic tool due to direct detection of molecular indicators from non-invasively collected samples as well as the potential for high-throughput analysis in a clinical setting; many studies have revealed the presence of viral peptides within easily accessed patient samples. However, evidence suggests that some viral peptides could serve as better indicators of COVID-19 infection status than others, due to potential misidentification of peptides derived from human host proteins, poor spectral quality, high limits of detection etc.MethodsIn this study we have compiled a list of 636 peptides identified from Sudden Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) samples, including from in vitro and clinical sources. These datasets were rigorously analyzed using automated, Galaxy-based workflows containing tools such as PepQuery, BLAST-P, and the Multi-omic Visualization Platform as well as the open-source tools MetaTryp and Proteomics Data Viewer (PDV).ResultsUsing PepQuery for confirming peptide spectrum matches, we were able to narrow down the 639-peptide possibilities to 87 peptides that were most robustly detected and specific to the SARS-CoV-2 virus. The specificity of these sequences to coronavirus taxa was confirmed using Unipept and BLAST-P. Through stringent p-value cutoff combined with manual verification of peptide spectrum match quality, 4 peptides derived from the nucleocapsid phosphoprotein and membrane protein were found to be most robustly detected across all cell culture and clinical samples, including those collected non-invasively.ConclusionWe propose that these peptides would be of the most value for clinical proteomics applications seeking to detect COVID-19 from patient samples. We also contend that samples harvested from the upper respiratory tract and oral cavity have the highest potential for diagnosis of SARS-CoV-2 infection from easily collected patient samples using mass spectrometry-based proteomics assays.

Development of the C12Im-Cl-assisted method for rapid sample preparation in proteomic application.

Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods used prior to MS, including protein extraction and digestion, remain bottlenecks in the large-scale clinical application of proteomics. Ionic liquids, composed entirely of ions, have high solubility in various solvents. In this study, the effects of the cationic surfactant 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) on protein digestion were evaluated for clinical proteomic applications. C12Im-Cl was compatible with trypsin and reduced the protein digestion time from 16 h to 1 h. Residual C12Im-Cl was easily removed with a strong anion exchange membrane before MS. We evaluated the performance of C12Im-Cl extraction and rapid protein digestion using formalin-fixed paraffin-embedded liver cancer tissues. The number of proteins and peptides identified was nearly equal to that identified by the traditional filter-aided sample preparation method (2705 vs. 2739 and 16 682 vs. 17 214). In general, the C12Im-Cl-aided rapid sample preparation method is promising for proteomic applications.

Proteomic approaches to investigate gammaherpesvirus biology and associated tumorigenesis.

The DNA viruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are members of the gammaherpesvirus subfamily, a group of viruses whose infection is associated with multiple malignancies, including cancer. The primary host for these viruses is humans and, like all herpesviruses, infection with these pathogens is lifelong. Due to the persistence of gammaherpesvirus infection and the potential for cancer formation in infected individuals, there is a driving need to understand not only the biology of these viruses and how they remain undetected in host cells but also the mechanism(s) by which tumorigenesis occurs. One of the methods that has provided much insight into these processes is proteomics. Proteomics is the study of all the proteins that are encoded by a genome and allows for (i) identification of existing and novel proteins derived from a given genome, (ii) interrogation of protein-protein interactions within a system, and (iii) discovery of druggable targets for the treatment of malignancies. In this chapter, we explore how proteomics has contributed to our current understanding of gammaherpesvirus biology and their oncogenic processes, as well as the clinical applications of proteomics for the detection and treatment of gammaherpesvirus-associated cancers.

The New and the Old: Platform Cross-Validation of Immunoaffinity MASS Spectrometry versus ELISA for PromarkerD, a Predictive Test for Diabetic Kidney Disease.

PromarkerD is a proteomics derived test for predicting diabetic kidney disease that measures the concentrations of three plasma protein biomarkers, APOA4, CD5L and IBP3. Antibodies against these proteins were developed and applied to a multiplexed immunoaffinity capture mass spectrometry assay. In parallel, and facilitating current clinical laboratory workflows, a standard ELISA was also developed to measure each protein. The performance characteristics of the two technology platforms were compared using a cohort of 100 samples, with PromarkerD test scores demonstrating a high correlation (R = 0.97). These technologies illustrate the potential for large scale, high throughput clinical applications of proteomics now and into the future.

Quantification of Biomarker Proteins Using Reverse-Phase Protein Arrays.

It is expected that antibody-based proteomics will soon occupy a pivotal position in the discovery and validation of biomarkers and therapeutic targets. The reverse-phase protein array (RPPA) is an antibody-based proteomic method that can quantify the expression of multiple posttranslationally modified proteins (such as those that have been phosphorylated) across a large number of protein samples. RPPA is highly sensitive and requires only very small protein samples. This feature, in combination with large antibody libraries, makes RPPA ideal for clinical proteomics, as well as the fact that it is an expandable multiplex assay. In Volume 14, Issue 1 of Proteomics Clinical Applications, Suzuki and colleagues report for the first time a study comparing RPPA and immunohistochemistry for quantification of seven biomarker proteins used for subtyping of diffuse large B-cell lymphoma. Such combination of multiple biomarkers is likely to increase diagnostic accuracy and can be used for precise classification of this heterogeneous disease.

Proteomic Research of Molecular Defects in Globozoospermia.

Globozoospermia is a form of teratozoospermia characterized by round-headed spermatozoa and lack of acrosome. These spermatozoa cannot penetrate the zona pellucida of the oocyte, resulting in unsuccessful fertilization and infertility. When intracytoplasmic sperm injection is performed, the fertilization rate tends to be low. Until now, the causes of this disorder remain to be elucidated; however, mutations of some genes segregating on an autosomal recessive mode have been associated with this infertile condition. DPY19L2 (dpy-19-like 2 [Caenorhabditis elegans]) codes for a transmembrane protein expressed predominantly in spermatids, with specific localization limited to the internal nuclear membrane. Genetic defects in the DPY19L2 gene have been demonstrated the most frequent genetic cause of globozoospermia; however, intracellular molecular pathways related to its encoded protein are largely unknown. In this issue of Proteomics Clinical Applications, Guo and co-workers investigate the proteome of gloobozoospermic spermatozoa. The authors identified 491 proteins that are differentially expressed in globozoospermia (370 are upregulated and 121 are downregulated in DPY19L2-deficient globozoospermic sperm). Notably, the molecular defects identified by the authors are closely related to biological processes involved in acrosome formation, chromatin composition, sperm-egg binding, and fertilization.

Apolipoprotein profiling as a personalized approach to the diagnosis and treatment of dyslipidaemia.

An elevated low-density lipoprotein cholesterol concentration is a classical risk factor for cardiovascular disease. This has led to pharmacotherapy in patients with atherosclerotic heart disease or high heart disease risk with statins to reduce serum low-density lipoprotein cholesterol. Even in patients in whom the target levels of low-density lipoprotein cholesterol are reached, there remains a significant residual cardiovascular risk; this is due, in part, to a focus on low-density lipoprotein cholesterol alone and neglect of other important aspects of lipoprotein metabolism. A more refined lipoprotein analysis will provide additional information on the accumulation of very low-density lipoproteins, intermediate density lipoproteins, chylomicrons, chylomicron-remnants and Lp(a) concentrations. Instead of measuring the cholesterol and triglyceride content of the lipoproteins, measurement of their apolipoproteins (apos) is more informative. Apos are either specific for a particular lipoprotein or for a group of lipoproteins. In particular measurement of apos in atherogenic particles is more biologically meaningful than the measurement of the cholesterol concentration contained in these particles. Applying apo profiling will not only improve characterization of the lipoprotein abnormality, but will also improve definition of therapeutic targets. Apo profiling aligns with the concept of precision medicine by which an individual patient is not treated as ‘average’ patient by the average (dose of) therapy. This concept of precision medicine fits the unmet clinical need for stratified cardiovascular medicine. The requirements for clinical application of proteomics, including apo profiling, can now be met using robust mass spectrometry technology which offers desirable analytical performance and standardization.

Development and Validation of Multiple Reaction Monitoring (MRM) Assays for Clinical Applications.

Selected/multiple reaction monitoring-mass spectrometry (SRM/MRM) is an analytical method that is frequently combined to the use of stable isotope-labeled standard (SIS) peptides for absolute protein quantification. The application of SRM/MRM is a relatively recent development in the proteomics field for analysis of biological samples (plasma, urine, cell/tissue lysates) targeting to a large extent biomarker validation. Although MRM generally by-passes the use of antibodies (being linked to sub-optimal assay specificity in many cases), bioanalytical validation of MRM protocols has not been robustly appliedbecause of sensitivity issues, in contrary to antibody-based methods. In this chapter, we will discuss the points that should be addressed for MRM method development in clinical proteomics applications.

Proteomics‐Based Investigations of Neglected and Tropical Diseases

Proteomics‐Based Investigations of Neglected and Tropical Diseases

Proteomic Analysis of Exosomes and Its Application in HIV-1 Infection.

Exosomes are 30-100nm extracellular vesicles secreted from late endosomes by various types of cells. Numerous studies have suggested that exosomes play significant roles in human immunodeficiency virus 1 (HIV-1) biogenesis. Proteomics coupled with exosome fractionation has been successfully used to identify various exosomal proteins and helped to uncover the interactions between exosomes and HIV-1. To inform the current progress in the intersection of exosome, proteomics, and HIV-1, this review is focused on: i) analyzing different exosome isolation, purification methods, and their implications in HIV-1 studies; ii) evaluating the roles of various proteomic techniques in defining exosomal contents; iii) discussing the research and clinical applications of proteomics and exosome in HIV-1 biology.

New Strategies and Challenges in Lung Proteomics and Metabolomics. An Official American Thoracic Society Workshop Report.

This document presents the proceedings from the workshop entitled, "New Strategies and Challenges in Lung Proteomics and Metabolomics" held February 4th-5th, 2016, in Denver, Colorado. It was sponsored by the National Heart Lung Blood Institute, the American Thoracic Society, the Colorado Biological Mass Spectrometry Society, and National Jewish Health. The goal of this workshop was to convene, for the first time, relevant experts in lung proteomics and metabolomics to discuss and overcome specific challenges in these fields that are unique to the lung. The main objectives of this workshop were to identify, review, and/or understand: (1) emerging technologies in metabolomics and proteomics as applied to the study of the lung; (2) the unique composition and challenges of lung-specific biological specimens for metabolomic and proteomic analysis; (3) the diverse informatics approaches and databases unique to metabolomics and proteomics, with special emphasis on the lung; (4) integrative platforms across genetic and genomic databases that can be applied to lung-related metabolomic and proteomic studies; and (5) the clinical applications of proteomics and metabolomics. The major findings and conclusions of this workshop are summarized at the end of the report, and outline the progress and challenges that face these rapidly advancing fields.

Proteomics: A new perspective for the understanding of pterygia.

Pterygia is a common ocular surface disease bothering both the patient because of its unsightly appearance and the surgeon because of its tendency to recur. The pathogenesis of pterygia is complex and the exact mechanism(s), especially at the molecular level, remains unknown. The use of modern proteomic techniques such as iTRAQ may yield new knowledge on the underlying pathogenesis of pterygia. In this issue of Proteomics Clinical Applications, Linghu etal. (article number 1600094) identified a total of 156 proteins that expressed differently between the pterygia and healthy conjunctiva using the isobaric tags for relative and absolute quantification based quantitative proteomic analysis. Most significantly, western blotting confirmed that two candidate proteins matrix metalloproteinase 10 (MMP-10) and CD34 were significantly upregulated in pterygia, suggesting that they have potential roles in the pathogenesis of pterygia. The findings in Linhu's study may provide a new perspective for the understanding of pterygia and develop a new therapeutic target.

Diving into the rat plasma proteome to get to the bottom of decompression sickness.

Decompression sickness (DCS) is the collective term for an array of signs and symptoms triggered by ambient pressure reduction. It is of particular concern to divers as they decompress on ascend from depth to sea surface, but despite a long history of studies the determinants of DCS risk are incompletely understood and there are no validated biomarkers. In this issue of Proteomics Clinical Applications, Lautridou etal. [8] report on their search for DCS biomarkers in rats exposed to simulated diving. By comparing the plasma proteomes from animals showing neurological symptoms to those emerging from dives unaffected, they identified several high-abundance proteins not previously associated with DCS. The most significant finding was a near depletion of thyroxine- and vitamin A transporter transthyretin in symptomatic rats. In addition to their potential role as diagnostic biomarkers, the proteins identified in Lautridou's study may offer new pieces in the yet incomplete puzzle of DCS etiology.

Proteomic Findings in Melanoma.

Although the emergence of proteomics as an independent branch of science is fairly recent, within a short period of time it has contributed substantially in various disciplines. The tool of mass spectrometry has become indispensable in the analysis of complex biological samples. Clinical applications of proteomics include detection of predictive and diagnostic markers, understanding mechanism of action of drugs as well as resistance mechanisms against them and assessment of therapeutic efficacy and toxicity of drugs in patients. Here, we have summarized the major contributions of proteomics towards the study of melanoma, which is a deadly variety of skin cancer with a high mortality rate.

Proteomics Clinical Applications Acknowledgements

PROTEOMICS – Clinical ApplicationsVolume 9, Issue 1-2 p. 253-254 Acknowledgements Proteomics Clinical Applications Acknowledgements First published: 06 February 2015 https://doi.org/10.1002/prca.201570016Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume9, Issue1-2Special Issue: Reviews 2015February 2015Pages 253-254 RelatedInformation

Application of clinical proteomics in acute respiratory distress syndrome.

Acute Respiratory Distress Syndrome (ARDS) is a devastating cause of hypoxic respiratory failure, which continues to have high mortality. It is expected that a comprehensive systems- level approach will identify global and complex changes that contribute to the development of ARDS and subsequent repair of the damaged lung. In the last decade, powerful genome-wide analytical and informatics tools have been developed, that have provided valuable insights into the mechanisms of complex diseases such as ARDS. These tools include the rapid and precise measure of gene expression at the proteomic level. This article reviews the contemporary proteomics platforms that are available for comprehensive studies in ARDS. The challenges of various biofluids that could be investigated and some of the studies performed are also discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-014-0034-1) contains supplementary material, which is available to authorized users.

Glycoproteome of human apolipoprotein A-I: N- and O-glycosylated forms are increased in patients with acute myocardial infarction

High-density lipoprotein (HDL) functionality, which is closely associated with its composition and transport capabilities, determines its role in atheroprotection. During acute phase processes, HDL seems to lose its anti-inflammatory and cytoprotective properties. In this study, we hypothesized that after an acute myocardial infarction apolipoprotein (Apo) A-I, the main protein component of HDL, might undergo changes in its molecular processing. Therefore, we have characterized theApo A-I proteome during the evolution of new-onset acute myocardial infarction (AMI). To this end, serum Apo A-I was characterized by 2-dimensional electrophoresis/mass-spectrometry in controls and AMI patients at admission (within the first 6hours after pain onset) and 8 hours, 16 hours, 24 hours, and 3days afterward. The Apo A-I glycoproteome was analyzed by lectin-based glycoprotein isolation methods and deglycosylation assays, and Apo A-I serum levels were evaluated by enzyme-linked immunosorbent assay (ELISA). The Apo A-I proteomic signature (5 spots: 28kDa/pI:5-5.75) was significantly altered in AMI patients 3days after the event with respect to controls. Increased levels of N- and O-glycosylated Apo A-I forms were found post-AMI. Apo A-I serum levels measured by ELISA were significantly changed and related to left ventricular ejection fraction, troponin-T, and C-reactive protein. The Apo A-I molecule measured by ELISA corresponded to the main glycosylated spots and was specifically O-GlcNAcylated in AMI patients. Therefore, our results demonstrate that Apo A-I is both N- and O-glycosylated and that there is an increase in Apo A-I glycosylation after AMI. Furthermore, the specific increase in the O-GlcNAcylated forms could have a relevant prognostic value and a protective role in the evolution of AMI.