Proteomics Mass Spectrometry Methods: Sample Preparation, Protein Digestion, and Research Protocols

Proteomics Mass Spectrometry Methods: Sample Preparation, Protein Digestion, and Research Protocols shares best practices collected across key laboratories and core facilities, taking the reader through key tactics for executing the most usual mass spectrometry experiments. Sections review research making use of MS proteomics experiments, focus on critical sample preparation, cover mammalian cell lines and samples from clinical tissue and biological fluids, discuss subcellular fractionation, provide methods for protein digestion both for in gel and in solution, and delve into key MS proteomics analysis protocols, including label-free LC-MS, TMT and iTRAQ labelled LC-MS, phosphorylation enrichment, ubiquination enrichment, and more. This book is the perfect lab manual for research teams or for use as a new staff training material. Core facility managers may also find it useful for sharing best practices with their staff and researchers.

• Explores the most common questions new researchers have
• Guides readers to properly design the workflow for successful integration of mass spectrometry into protein biochemical analyses
• Provides examples of sample preparation for a number of different materials, mammalian cells, and others

• Language: English
• Publisher: Academic Press
• Release date: Feb 13, 2024
• ISBN: 9780323906524

Book preview

Section I

Introduction

Outline

Chapter 1. Book introduction

Chapter 2. Introduction to sample preparation for proteomics and mass spectrometry

Chapter 1: Book introduction

Paula Meleady     School of Biotechnology, Dublin City University, Glasnevin, Dublin, Ireland

Abstract

Over the last decade, mass spectrometry–based proteomics has gained increased attention due to advances in sample preparation methods and instrumentation as well as improved protein databases. These advancements have led to the analysis of thousands of proteins in a sample and have made it possible to study global protein dynamics from cells to tissues and organisms. Proteins are the primary functional molecules in our cells and are responsible for almost all biological and cellular processes within the cell. In addition, the relationship between messenger RNA and protein expression is complex and not always correlated with each other for most genes and can vary significantly from protein to protein (Payne, 2015; Buccitelli and Selbach, 2020; Upadhya and Ryan, 2022 Jiang et al., 2023). Proteins are also subject to a diverse array of consequential post-translational modifications (PTMs) (e.g., phosphorylation, methylation, glycosylation, ubiquitination, etc.) which are not encoded by mRNA sequence or captured by transcript abundance. Therefore, understanding how protein abundances and their PTMs vary between healthy and disease states can provide insights into how biological activities are altered in disease conditions, thus potentially identifying new biomarkers of disease and novel therapeutic targets to improve patient outcomes.

Keywords

Biomarkers; Mass spectrometry; Peptides; Protein; Proteomics

Over the last decade, mass spectrometry (MS)–based proteomics has gained increased attention due to advances in sample preparation methods and instrumentation as well as improved protein databases. These advancements have led to the analysis of thousands of proteins in a sample and have made it possible to study global protein dynamics from cells to tissues and organisms. Proteins are the primary functional molecules in our cells and are responsible for almost all biological and cellular processes within the cell. In addition, the relationship between messenger RNA and protein expression is complex and not always correlated with each other for most genes and can vary significantly from protein to protein [1–4]. Proteins are also subject to a diverse array of consequential posttranslational modifications (PTMs) (e.g., phosphorylation, methylation, glycosylation, ubiquitination, etc.) which are not encoded by mRNA sequence or captured by transcript abundance. Therefore, understanding how protein abundances and their PTMs vary between healthy and disease states can provide insights into how biological activities are altered in disease conditions, thus potentially identifying new biomarkers of disease and novel therapeutic targets to improve patient outcomes.

The main idea of this methods book is to create a resource or starter guide for the non-expert proteomics scientist, as they apply proteomics techniques to answer a biological question. This book contains a range of methods' chapters from various experts in sample preparation for proteomics analysis. We have also shared some of the day-to-day protocols we use in our Core Proteomics Lab in Dublin City University, Dublin, Ireland. These methods can be used as a starting point for a user's own proteomics projects and can be optimized or modified to better suit the application being studied.

This book is divided into four main sections. Section I (Chapters 1 and 2) are introductory chapters to the book. Chapter 2 is a broad overview of proteomics and is a useful starting guide to understand the various aspects to a proteomics experiment from the initial sample preparation to analysis by mass spectrometry. Section II (Chapters 3–8) covers a range of protocols for the extraction of proteins from various biological samples, followed by methods for proteolytic digestion to peptides for mass spectrometry analysis. Chapter 3 describes a protocol for the extraction of membrane and membrane-associated proteins from mammalian cell lines and can be applied to both adherent and suspension cultured cells. Proteolytic digestion of proteins to peptides prior to LC-MS/MS analysis is described using two approaches, the filter-aided sample preparation method [5] and using a simplified kit-based approach. Chapter 4 describes a comprehensive method for the extraction of proteins from tissue samples, specifically from human muscle biopsy samples. This chapter also includes extensive guidelines on the removal of interfering chemicals from the peptide fractions prior to LC-MS/MS analysis. Chapter 5 describes a straightforward methodology for the preparation of a serum sample from a patient's blood. The method includes the use of an immunodepletion approach to remove high abundant proteins (e.g., IgG and serum albumin) from the serum sample to improve the detection of lower abundant proteins by LC-MS/MS. Chapter 6 describes methodologies for the preparation of both fungal and bacterial samples for proteomic analysis. Specifically, methods are described for sample preparation from the bacteria, Pseudomonas aeruginosa and Staphylococcus aureus, and from the fungi, Candida albicans and Aspergillus fumigatus, all of which are significant pathogens of humans. Chapter 7 describes a comprehensive method for the extraction and preparation of extracellular vesicles from urine for protein identification. Extracellular vesicles are challenging to extract from biological fluids [6]; however, have they have attracted a lot of interest in the clinical setting due to their potential to provide diagnostic information from liquid biopsies (e.g., urine, blood) in many disease indications including cancer, cardiovascular disease, and kidney disease [7–10]. Chapter 8 describes methods for protein extraction from animal samples, including information on the collection of exudates from postmortem muscle tissue.

Section III contains a protocol (Chapter 9) for in-gel digestion of proteins into peptides, with some modifications from the original published protocol [11]. This protocol is extremely useful for the extraction of protein samples from SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis) gels, followed by proteolytic digestion in-gel to produce peptides for mass spectrometry analysis. The method is used extensively to identify and quantify the components of protein complexes fractionated by native PAGE and in immunoprecipitation experiments using SDS-PAGE.

The final section, Section IV, includes a number of protocols and approaches to analyze the extracted peptide samples for protein identification and to identify differentially expressed proteins between experimental groups within the proteomic study being evaluated. Chapters 10 and 11 include informative protocols to identify differentially expressed proteins using both label-free and labeled (i.e., tandem mass tags) LC-MS/MS proteomic profiling and include advantages and disadvantages to both approaches. This book also includes protocols for the enrichment of common PTMs such as phosphorylation (Chapter 12) and ubiquitination (Chapter 13). The methods described in both chapters allow the site of modification to be characterized and identified, and the methods used are also amenable to differential phosphoproteomic and differential ubiquitinated proteomic studies to be carried out between experimental groups. Finally, Chapter 14 describes a high-throughput proteomics platform with the ability to analyze proteins rapidly and accurately for deep proteome coverage. It consists of an automated sample preparation press combined with an automated liquid chromatographic system, coupled with high-mass-accuracy mass spectrometry. Chapter 14 also includes a data-independent acquisition strategy for protein quantitation analysis, while Chapter 10 uses a data-dependent acquisition strategy for protein quantitation.

We are in very exciting times for the application of proteomics to understand many diseases and their progression, particularly with the development and recent launch (June 2023) of next generation high-performance mass spectrometers such as the Orbitrap Astral [12] and the TimsTOF Ultra (Bruker). ¹ These instruments are capable of faster throughput, deeper coverage, and higher sensitivity with accurate and precise quantitation, compared to existing instruments on the market. These advances in MS instrumentation functionalities and sensitivity have the potential to transform our understanding of disease phenotypes to identify new biomarkers and therapeutic targets of disease, and in particular diseases where there are unmet needs.

References

1. Payne S.H. The utility of protein and mRNA correlation. Trends Biochem Sci. 2015;40:1–3. doi: 10.1016/j.tibs.2014.10.010.

2. Buccitelli C, Selbach M. mRNAs, proteins and the emerging principles of gene expression control. Nat Rev Genet. 2020;21:630–644. doi: 10.1038/s41576-020-0258-4.

3. Upadhya S.R, Ryan C.J. Experimental reproducibility limits the correlation between mRNA and protein abundances in tumor proteomic profiles. Cell Rep Methods. 2022;2:100288 doi: 10.1016/j.crmeth.2022.100288.

4. Jiang D, Cope A.L, Zhang J, Pennell M. On the decoupling of evolutionary changes in mRNA and protein levels. Mol Biol Evol. 2023;40:msad169 doi: 10.1093/molbev/msad169.

5. Wiśniewski J.R, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6:359–362. doi: 10.1038/nmeth.1322.

6. Allelein S, Medina-Perez P, Lopes A.L.H, Rau S, Hause G, Kölsch A, et al. Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations. Sci Rep. 2021;11:11585 doi: 10.1038/s41598-021-91129-y.

7. Sun I.O, Lerman L.O. Urinary extracellular vesicles as biomarkers of kidney disease: from diagnostics to therapeutics. Diagnostics. 2020;10:311. doi: 10.3390/diagnostics10050311.

8. Martin-Ventura J.L, Roncal C, Orbe J, Blanco-Colio L.M. Role of extracellular vesicles as potential diagnostic and/or therapeutic biomarkers in chronic cardiovascular diseases. Front Cell Dev Biol. 2022;10:813885 doi: 10.3389/fcell.2022.813885.

9. . Jia E, Ren N, Shi X, Zhang R, Yu H, et al. Extracellular vesicle biomarkers for pancreatic cancer diagnosis: a systematic review and meta-analysis. BMC Cancer. 2022;22:573. doi: 10.1186/s12885-022-09463-x.

10. Lee Y, Ni J, Beretov J, Wasinger V.C, Graham P, Li Y. Recent advances of small extracellular vesicle biomarkers in breast cancer diagnosis and prognosis. Mol Cancer. 2023;22:33. doi: 10.1186/s12943-023-01741-x.

11. Shevchenko A, Tomas H, Havli J, Olsen J.V, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1:2856–2860. doi: 10.1038/nprot.2006.468.

12. Heil L.R, Damoc E, Arrey T.N, Pashkova A, Denisov E, et al. Evaluating the performance of the Astral mass analyzer for quantitative proteomics using data-independent acquisition. J Proteome Res. 2023 doi: 10.1021/acs.jproteome.3c00357.

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¹   https://www.bruker.com/en/news-and-events/news/2023/bruker-launches-timstof-ultra-with-csi-ultra.html

Chapter 2: Introduction to sample preparation for proteomics and mass spectrometry

Michael Henry ¹ , and Paula Meleady ¹ , ²       ¹ National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin, Ireland      ² School of Biotechnology, Dublin City University, Glasnevin, Dublin, Ireland

Abstract

Proteomics is a complex field of study which involves the application of analytical chemistry, molecular biology, biochemistry, and genetics to the analysis of the structure, function, and interactions of proteins of a particular cell, tissue, or organism. Proteins are the principal functional molecules within a cell and are therefore more likely to reflect the phenotype of any living material. Quantitative proteomics in disease phenotypes such as cancer can facilitate the understanding the mechanisms of disease development and progression, and discovering biomarkers for disease diagnosis and new therapeutic targets of disease. This review gives a broad overview of the various steps required to analyze and characterize the proteome of a biological system, from the initial sample preparation to liquid chromatography mass spectrometry (LC-MS) analysis and finally to data analysis in order to reveal important insights into the data in a biological context.

Keywords

In solution digestion; Isotope labels; iTRAQ; Label-free proteomics; Mass spectrometry; Proteomics; Targeted LC-MS/MS; TMT

1. Introduction

Proteomics is a complex field of study which involves the application of analytical chemistry, molecular biology, biochemistry, and genetics to analyzing the structure, function, and interactions of proteins of a particular cell, tissue, or organism. Proteins are the principal functional molecules within a cell and are therefore more likely to reflect the phenotype of any living material. The inherent complexity of the proteome must also take into account that proteins are subject to post-translational modifications, trafficking, changes in subcellular location, protein–protein interactions, and complex formation, all contributing to biological function [1]. The importance of studying the proteome of a biological sample is demonstrated by the fact that the analysis of mRNA expression profiles is not necessarily a direct reflection of the protein expression profile of the cell [2]. As a result, many studies have shown a lack of correlation between mRNA and protein expression levels [ 3–8 ].

The analysis of the whole protein, typically referred to as top-down proteomics, is the mass analysis of an intact protein or its isoforms [9,10]. For example, within the biopharmaceutical industry whole protein analysis is required to ensure biotherapeutics are structurally defined and reproduced identically between production runs [11]. Bottom-up proteomics involves the identification of proteins through partial characterization of their amino acid sequence following proteolytic enzyme digestion of the intact proteins prior to mass spectrometry analysis. By comparing the masses of the proteolytic peptides or their mass spectra with those predicted from a theoretically digested sequence database, peptides can be identified and multiple peptide sequences assembled into a protein identification.

Proteomics relies on three basic technological cornerstones which include (1) a method to extract and separate complex protein or peptide mixtures, (2) mass spectrometry to acquire the data necessary to identify individual proteins, and (3) bioinformatics to analyze and assemble the MS data.

2. Sample preparation for proteomic analysis

Due to the complexity of the proteome, there is no one standard method for preparing protein samples for analysis by proteomics [12]. Protein extraction is the first step of sample preparation for proteomics and includes methodologies based on detergent-based lysis, organic solvents, sample disruption by sonication, freeze/thaw cycles, mechanical disruption, or combinations of these methods. Common lysis buffer components include denaturants (e.g., urea and thiourea), ionic detergents (e.g., SDS), and nonionic detergents (Triton X-100, NP-40) to lyse cells and solubilize proteins. Proteomic protocols vary depending on the sample number, type, experimental goals, and analysis method used. Many factors are considered when designing sample preparation strategies, which can include the sample source, type, physical properties, sample abundance, complexity, and cellular location of the proteins. Each technique has its own advantage and disadvantage. For example, the use of detergents is well documented for protein extraction efficiency; however, their presence down stream of sample preparation can cause issues when using liquid chromatography and with MS instruments. Sample preparation methods such as filter aided sample preparation (FASP) [13] and suspension trapping (S-trap) [14] can effectively remove the surfactant sodium dodecyl sulfate (SDS) from extracted protein samples. Alternatives to FASP and S-trap techniques when sample starting material is minimal hence requiring maximal proteome coverage and minimal sample loss, are solid-phase enhanced separation preparation (SP3) and in-Stage Tip (iST) techniques [15]. It must be noted that iST is not compatible with SDS [16]. There are also MS-compatible, commercially available detergents that are widely used, including Rapigest (Waters), ProteaseMax (Promega), and Invitrosol (Thermo), which degrade with the addition of heat or acidic pH conditions.

Workflows that incorporate optimized cellular lysis, subcellular fractionation, depletion of high-abundance proteins, or enrichment of select proteins can all contribute to the accurate identification and quantitation of protein samples. Enrichment and/or fractionation steps can be introduced at the protein and/or peptide level if sample complexity needs to be reduced or when a specific subset of proteins/peptides are of interest. In all cases, the quality and reproducibility of sample extraction and preparation can significantly impact the research