LC–MS Based Detection of Differential Protein Expression

Abstract

While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics. Significant technical advances at both sample preparation/separation and mass spectrometry levels have revolutionized comprehensive proteome analysis. Moreover, automation and robotics for sample handling process permit multiple sampling with high throughput.For LC-MS based quantitative proteomics, sample preparation turns out to be critical step, as it can significantly influence sensitivity of downstream analysis. Several sample preparation strategies exist, including depletion of high abundant proteins or enrichment steps that facilitate protein quantification but with a compromise of focusing on a smaller subset of a proteome. While several experimental strategies have emerged, certain limitations such as physiochemical properties of a peptide/protein, protein turnover in a sample, analytical platform used for sample analysis and data processing, still imply challenges to quantitative proteomics. Other aspects that make analysis of a proteome a challenging task include dynamic nature of a proteome, need for efficient and fast analysis of protein due to its constant modifications inside a cell, concentration range of proteins that exceed dynamic range of a single analytical method, and absence of appropriate bioinformatics tools for analysis of large volume and high dimensional data. This paper gives an overview of various LC-MS methods currently used in quantitative proteomics and their potential for detecting differential protein expression. Fundamental steps such as sample preparation, LC separation, mass spectrometry, quantitative assessment and protein identification are discussed.For quantitative assessment of protein expression, both label and label free approaches are evaluated for their set of merits and demerits. While most of these methods edge on providing "relative abundance" information, absolute quantification is achieved with limitation as it caters to fewer proteins. Isotope labeling is extensively used for quantifying differentially expressed proteins, but is severely limited by successful incorporation of its heavy label. Lengthy labeling protocols restrict the number of samples that can be labeled and processed. Alternatively, label free approach appears promising as it can process many samples with any number of comparisons possible but entails reproducible experimental data for its application.

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