[Paper Review] Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry

Yuming Jiang, Rex Devasahayam Arokia Balaya+17 more|PubMed|Nov 13, 2023

Advanced Proteomics Techniques and Applications765 references8 citations

TL;DR

This paper provides a comprehensive overview of bottom-up proteomics workflows using mass spectrometry, spanning from biochemistry basics to data interpretation and validation.

ABSTRACT

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics.

Motivation & Objective

• Motivate the study of bottom-up proteomics as a versatile approach for proteome analysis.
• Summarize the range of proteomics methods used to identify and quantify proteins and proteoforms.
• Provide guidance for novices and experienced researchers on workflow differences and practical considerations.
• Bridge concepts from biochemistry to data interpretation and orthogonal validation in shotgun proteomics.

Proposed method

• Describe biochemistry basics and protein extraction relevant to bottom-up proteomics.
• Outline sample preparation and digestion strategies for generating peptides.
• Summarize mass spectrometry approaches and data acquisition considerations for peptide analysis.
• Discuss data interpretation, protein identification/quantification, and proteoform characterization.
• Highlight post-translational modifications, protein-protein interactions, and stability assessments.
• Present orthogonal validation techniques and practical guidance for novices and experts.

Experimental results

Research questions

• RQ1 What are the dominant bottom-up (shotgun) proteomics workflows used in modern studies?
• RQ2 How do proteomics strategies differ across objectives such as simple identification, quantification, PTMs, and proteoforms?
• RQ3 What practical guidance and considerations help novices adopt shotgun proteomics workflows?
• RQ4 What are common validation approaches to corroborate proteomics findings?

Key findings

• The work provides a comprehensive overview of diverse proteomics methods from biochemistry to biological interpretation.
• It covers protein extraction, digestion, and peptide-level analysis essential for bottom-up workflows.
• It explains strategies for absolute and relative quantification, PTMs, and proteoform analysis.
• It discusses protein-protein interactions, structural alterations, and protein stability within bottom-up frameworks.
• It emphasizes orthogonal validation and practical guidance to aid practitioners new to shotgun proteomics.