Challenges and developments in protein identification using mass spectrometry

• Protein-identification strategies using mass spectrometry.
• Challenges in identification of proteoforms.
• “Bottom-up”, “middle-down” and “top-down” proteomics workflows.
• Developments in mass-spectrometric fragmentation instrumentation and algorithms.

Mass-spectrometry (MS)-based proteomics is the most powerful approach for identifying proteins and determining protein expression in tissues under different conditions to identify post-translational modifications in response to stimuli and to characterize protein interactions. Protein identification is a key step in characterizing proteomes to describe biological processes and to discover disease-related biomarkers, pharmaceutical targets, protein functions or interactions. In all proteomics workflows, whether commonly-applied gel-electrophoresis-based methods or gel-free approaches, MS is an indispensable tool for the identification of protein sequences and modifications. The complexity, high abundance, dynamic range, presence of similar proteins, and several forms of the same protein all raise challenges for analytical instrumentation and data-analysis software. This review provides an introduction to key terms, methods and challenges in protein identification, and summarizes current solutions and trends, including novel data-collection approaches, bioinformatics and instrumentation developments.