Functionalizing tandem mass tags for streamlining click-based quantitative chemoproteomics

Mass spectrometry-based quantitative chemoproteomics is crucial for functional biology and drug discovery. It aims to identify protein targets and specific residues modified by covalent chemical probes. Current chemoproteomics platforms focus on improving covalent labeling chemistries, sample preparation workflows, and data acquisition speed. Significant progress has been made in developing diverse covalent labeling chemistries targeting various amino acid side chains, with cysteine residues remaining a focus due to their functional roles and druggability. Improvements in sample preparation, such as single-pot, solid-phase enhanced sample preparation (SP3), and data analysis software like pLink, MSFragger, and SAGE, have increased coverage and reduced processing time. Isobaric labeling, using reagents like iTRAQ and TMT, significantly enhances data acquisition speed by multiplexing samples. However, the conventional late-stage isobaric labeling after proteolytic digestion increases sample-to-sample variance and prolongs processing. Alternative strategies, like the silane-based cleavable isotopically labeled proteomics (sCIP) and azidoTMT methods, introduce isobaric labels earlier, improving efficiency. The sCIP platform, although limited by 6-plex multiplexing, paved the way for the current work. AzidoTMT achieved 11-plex multiplexing, showing improved coverage and reduced variance but relied on antibody-based enrichment with limitations for TMTPro reagents. This paper addresses the need for a robust, easily implementable enrichment-based isobaric labeling method by introducing the sCIP-TMT platform.

The paper reviews the advancements in chemoproteomics, focusing on three key areas: covalent labeling chemistries, sample preparation, and data acquisition. It details the development of various covalent labeling chemistries targeting different amino acids, emphasizing the continued interest in cysteine modifications for drug development. The literature review highlights progress in sample preparation methods, including SP3, and advancements in data analysis software for faster processing. The use of isobaric labeling techniques, such as TMT and iTRAQ, for multiplexing samples and increasing data acquisition speed is discussed. Existing challenges related to late-stage isobaric labeling and the limitations of antibody-based enrichment in previous methods are also addressed, leading to the introduction of the sCIP-TMT platform as a potential solution to these challenges.

The sCIP-TMT platform was developed using a custom-synthesized sCIP-Gly-NH2 reagent. This reagent was synthesized via solid-phase peptide synthesis (SPPS) and contains biotin, a cleavable dialkoxydiphenylsilane (DADPS) group, an azide for click chemistry, and a free N-terminus amine for conjugation with commercially available TMT reagents. The synthesis and high yield (>99% conversion) of the sCIP-TMT reagents were confirmed using liquid chromatography-mass spectrometry (LC-MS). For cysteine profiling, HEK293T cell lysates were labeled with iodoacetamide alkyne (IAA), followed by click conjugation with the sCIP-TMT reagent. After proteolytic digestion, samples were enriched using streptavidin, the DADPS linker cleaved, and peptides eluted. LC-MS/MS analysis identified proteins, peptides, and cysteines. Diagnostic ion mining analysis identified the TMT reporter ion (m/z 126.1277) and a cysteine desulfurization ion (m/z 668.3896) as dominant fragments. Collision energy ramping determined optimal parameters for MS2-based TMT experiments. The methodology was extended to a 10-plex TMT set for high-throughput cysteine chemoproteomics and accurate quantification of cysteine peptide abundance using spike-in analysis with varying sample ratios (1:1 and 1:5:10:15). FAIMS-MS2 and SPS-MS3 analyses were used to assess quantification fidelity. The sCIP-TMT method was compared to a traditional IA-DTB/TMT workflow, evaluating coefficient of variance and peptide coverage. Finally, a screen with four cysteine-reactive electrophiles was conducted to assess the platform's compatibility with fragment electrophile screening, followed by comparative analysis to establish known and novel ligandable cysteines and assessment of the platform for N-terminal peptide labeling using ethynyl-2PCA labeling.

The sCIP-TMT platform demonstrated high efficiency in synthesizing sCIP-TMT reagents with >99% conversion. Using sCIP-TMTzero, high proteomic coverage was achieved, identifying 3856 proteins, 11,219 peptides, and 8543 unique cysteines. The TMTzero reporter ion was the dominant ion in nearly 100% of modified spectra. A cysteine desulfurization ion (m/z 668.3896) was also identified. Optimal collision energy for MS2 analysis was determined to be 35%. The sCIP-TMT10plex platform exhibited high overall proteomic coverage, comparable to previous studies, with an estimated 6h reduction in sample preparation time. Accurate quantification of cysteine peptide abundance was achieved in spike-in experiments. Compared to the IA-DTB/TMT workflow, sCIP-TMT showed reduced sample-to-sample variance (5.2% and 7.8% median coefficient of variance for 1:1 and 1:4 ratios, respectively, versus 9.6% and 9.7% for IA-DTB/TMT). A screen using four cysteine-reactive electrophiles identified 789 high-confidence liganded cysteines. The platform showed high concordance with previous studies on known ligandable cysteines and also identified novel ligandable cysteines. Furthermore, the sCIP-TMT platform proved compatible with N-terminal peptide labeling using ethynyl-2PCA, identifying over 700 unique peptide N-termini.

The sCIP-TMT platform addresses the limitations of existing isobaric labeling methods by enabling early sample pooling, reducing sample-to-sample variance and preparation time. The high accuracy of quantification and high coverage achieved in the study validate its efficacy. The compatibility with fragment electrophile screening broadens its applicability. The identification of both known and novel ligandable cysteines highlights its potential for drug discovery. The identification of the desulfurization ion as a characteristic fragment for modified cysteine peptides enhances confidence in results and opens opportunities for future improvements in differentiating false positive identifications. The success of the platform with N-terminal labeling using ethynyl-2PCA expands its potential applications.

The sCIP-TMT platform offers a significant advancement in chemoproteomics, enabling high-throughput, high-coverage cysteine profiling with reduced sample preparation time and improved quantification accuracy. Its compatibility with various applications, including fragment screening and N-terminal proteomics, makes it a versatile tool for studying protein-small molecule interactions. Future directions include expanding multiplexing capabilities beyond 10-plex and exploring its application to other amino acid residues.

The sCIP-Gly-NH2 reagent was obtained in a moderate 53% yield, which could be improved. The current approach might not be readily amenable to established automation workflows due to early sample pooling. However, this could be addressed by adapting the workflow to 96-well plate formats.