Advances in quantitative high‐throughput phosphoproteomics with sample multiplexing

Abstract

Eukaryotic protein phosphorylation modulates nearly every major biological process.Phosphorylation regulates protein activity, mediates cellular signal transduction, and manipulates cellular structure. Consequently, the dysregulation of kinase and phosphatase pathways has been linked to a multitude of diseases. Mass spectrometrybased proteomic techniques are increasingly used for the global interrogation of perturbations in phosphorylation-based cellular signaling. Strategies for studying phosphoproteomes require high-specificity enrichment, sensitive detection, and accurate localization of phosphorylation sites with advanced LC-MS/MS techniques and downstream informatics. Sample multiplexing with isobaric tags has also been integral to recent advancements in throughput and sensitivity for phosphoproteomic studies.Each of these facets of phosphoproteomics analysis present distinct challenges and thus opportunities for improvement and innovation. Here, we review current methodologies, explore persistent challenges, and discuss the outlook for isobaric tag-based quantitative phosphoproteomic analysis.

Progress in methodologies and quality‐control strategies in protein cross‐linking mass spectrometry

“…The SCX fractionation method can further be customized using StageTips [106]. Other fractionation methods such as hydrophilic strong anionic exchange (hSAX) chromatography [70] and hydrophilic interaction chromatography (HILIC) [91], which are commonly employed to increase the depth of proteome coverage and phospho‐peptides enrichment [107], can also be utilized for enrichment cross‐linked peptides [38, 108]. Furthermore, two‐dimensional peptide separation has been shown to be efficient in recent studies [70, 109, 110].…”

Insights Into the Biogenesis and Emerging Functions of Lipid Droplets From Unbiased Molecular Profiling Approaches

“…Reflecting the increasing complexity found in combinatorial PTM-driven signaling and proteome regulation ( Aggarwal et al, 2021 ), recent studies confirm that LD proteins are substrates for, and/or regulators of, acetylation ( Qian et al, 2017 ; Nguyen et al, 2019 ), palmitoylation ( Suzuki et al, 2015 ), ubiquitylation ( Zhang et al, 2018 ; BasuRay et al, 2019 ; Sugihara et al, 2019 ), ISGylation ( Thery et al, 2021 ), or UFMylation ( Eck et al, 2020 ). Proteomics procedures hold a key to globally understand the dynamics and function of these PTMs, but a majority of strategies have relied on the affinity purification of peptides bearing a specific PTM before its analysis by mass spectrometry ( Murray et al, 2019 ; Nelson et al, 2021 ; Paulo and Schweppe, 2021 ; Rivera et al, 2021 ; Hsieh et al, 2022 ). These procedures have enabled the charting of signaling networks and their dynamics with unprecedented resolution, but they are limited to describe the relationships and interplay of different PTMs among each other, and often require substantial sample amounts.…”

Advances in proteomics sample preparation and enrichment for phosphorylation and glycosylation analysis

“…MS technology is emerging as the most powerful method for comprehensive analysis of protein phosphorylation and glycosylation levels due to its immediate, high-throughput, excellent specificity, and high sensitivity [9][10][11]. However, direct studies of PTMs proteins by MS are susceptible to interference from complex matrices due to the low concentration of PTMs proteins in the sample.…”