Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation

Nakayasu, Ernesto; Gritsenko, Marina; Piehowski, Paul; Gao, Yuqian; Orton, Daniel J.; Schepmoes, Athena; Fillmore, Thomas; Frohnert, Brigitte I.; Rewers, Marian; Krischer, Jeffrey P.; Ansong, Charles; Suchy‐Dicey, Astrid; Evans‐Molina, Carmella; Qian, Weijun; Webb‐Robertson, Bobbie‐Jo; Metz, Thomas O.

doi:10.1038/s41596-021-00566-6

Cited by 128 publications

(101 citation statements)

References 159 publications

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“…To improve current diagnosis and therapeutic responses in SS patients, better characterization and understanding of the disease is urgently needed. With a coherent pipeline connecting potential biomarker identification with well-established methods for validation, mass spectrometry-based approaches are an effective way to discover novel biomarkers ( Rifai et al, 2006 ; Aebersold and Mann, 2016 ; Nakayasu et al, 2021 ). Identifying protein biomarkers in tear washes and saliva offers several advantages as it is manageable for sampling/repeated sampling in addition to being inexpensive and non-invasive ( Chen et al, 2015 ; Nakayasu et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

“…With a coherent pipeline connecting potential biomarker identification with well-established methods for validation, mass spectrometry-based approaches are an effective way to discover novel biomarkers ( Rifai et al, 2006 ; Aebersold and Mann, 2016 ; Nakayasu et al, 2021 ). Identifying protein biomarkers in tear washes and saliva offers several advantages as it is manageable for sampling/repeated sampling in addition to being inexpensive and non-invasive ( Chen et al, 2015 ; Nakayasu et al, 2021 ). Here, we performed an unbiased investigation using quantitative proteomics of SS saliva and tear washes compared to healthy controls.…”

Section: Discussionmentioning

confidence: 99%

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Proteomics Analysis of Tears and Saliva From Sjogren’s Syndrome Patients

et al. 2021

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Sjogren’s syndrome (SS) is characterized by dysfunctional mucous membranes and dysregulated moisture-secreting glands resulting in various symptoms, including dry mouth and dry eyes. Here, we wanted to profile and compare the tear and saliva proteomes of SS patients to healthy controls. Tear and saliva samples were collected and subjected to an isotopic dimethylation labeling shotgun proteomics workflow to identify alterations in protein levels. In tear samples, we identified 83 upregulated and 112 downregulated proteins. Pathway enrichment analysis of the changing proteins by Metascape identified leukocyte transendothelial migration, neutrophil degranulation, and post-translation protein phosphorylation in tears of SS patients. In healthy controls’ tears, an enrichment for proteins related to glycolysis, amino acid metabolism and apoptotic signaling pathway were identified. In saliva, we identified 108 upregulated and 45 downregulated proteins. Altered pathways in SS patients’ saliva included cornification, sensory perception to taste and neutrophil degranulation. In healthy controls’ saliva, an enrichment for proteins related to JAK-STAT signaling after interleukin-12 stimulation, phagocytosis and glycolysis in senescence were identified. Dysregulated protease activity is implicated in the initiation of inflammation and immune cell recruitment in SS. We identified 20 proteases and protease inhibitors in tears and 18 in saliva which are differentially expressed between SS patients and healthy controls. Next, we quantified endogenous proteoglycan 4 (PRG4), a mucin-like glycoprotein, in tear wash and saliva samples via a bead-based immune assay. We identified decreased levels of PRG4 in SS patients’ tear wash compared to normal samples. Conversely, in saliva, we found elevated levels of PRG4 concentration and visualized PRG4 expression in human parotid gland via immunohistological staining. These findings will improve our mechanistic understanding of the disease and changes in SS patients’ protein expression will help identify new potential drug targets. PRG4 is among the promising targets, which we identified here, in saliva, for the first time.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Proteomics Analysis of Tears and Saliva From Sjogren’s Syndrome Patients

et al. 2021

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“…[42][43][44] Peptide prefractionation or enrichment Enrichment methods based on affinity binding require large starting protein amounts. [18,45,46] For example, sample fractionation or depletion is commonly required before tandem mass spectroscopy (MS/MS) analysis regardless of the type of body fluids because of the proteome complexity or the presence of high abundance proteins in body fluids [12,27]. In the case of blood, the dynamic ranges differ by a factor of 10 10 between serum albumin (35-50 mg/mL in normal conditions), the most abundant protein, and cytokines (low pg/mL range).…”

Section: Efficiency Of Protein Preparationmentioning

confidence: 99%

“…A detailed comparison of these methods is beyond the scope of this paper. However, several studies have described the differences among such methods [18,42,43,46,49,53].…”

Section: Efficiency Of Protein Preparationmentioning

confidence: 99%

Review of Liquid Chromatography-Mass Spectrometry-Based Proteomic Analyses of Body Fluids to Diagnose Infectious Diseases

Hayoung

Kim

2022

IJMS

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Rapid and precise diagnostic methods are required to control emerging infectious diseases effectively. Human body fluids are attractive clinical samples for discovering diagnostic targets because they reflect the clinical statuses of patients and most of them can be obtained with minimally invasive sampling processes. Body fluids are good reservoirs for infectious parasites, bacteria, and viruses. Therefore, recent clinical proteomics methods have focused on body fluids when aiming to discover human- or pathogen-originated diagnostic markers. Cutting-edge liquid chromatography–mass spectrometry (LC-MS)-based proteomics has been applied in this regard; it is considered one of the most sensitive and specific proteomics approaches. Here, the clinical characteristics of each body fluid, recent tandem mass spectroscopy (MS/MS) data-acquisition methods, and applications of body fluids for proteomics regarding infectious diseases (including the coronavirus disease of 2019 [COVID-19]), are summarized and discussed.

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“…Until now several reviews were published devoted to practical applications of MRM and parallel reaction monitoring (PRM) in system biology and biomedical research [7][8][9][10][11][12]. This review is dedicated to targeted proteomics, including design of MRM experiments, with special focus on sample preparation pitfalls and artifacts, analysis of experimental data, and recently improved MRM-based strategies.…”

Section: Introductionmentioning

confidence: 99%

Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

2021

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Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is now the main analytical method for the identification and quantification of peptides and proteins in biological samples. In modern research, identification of biomarkers and their quantitative comparison between samples are becoming increasingly important for discovery, validation, and monitoring. Such data can be obtained following specific signals after fragmentation of peptides using multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) methods, with high specificity, accuracy, and reproducibility. In addition, these methods allow measurement of the amount of posttranslationally modified forms and isoforms of proteins. This review article describes the basic principles of MRM assays, guidelines for sample preparation, recent advanced MRM-based strategies, applications and illustrative perspectives of MRM/PRM methods in clinical research and molecular biology.

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Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation

Cited by 128 publications

References 159 publications

Proteomics Analysis of Tears and Saliva From Sjogren’s Syndrome Patients

Proteomics Analysis of Tears and Saliva From Sjogren’s Syndrome Patients

Review of Liquid Chromatography-Mass Spectrometry-Based Proteomic Analyses of Body Fluids to Diagnose Infectious Diseases

Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

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