Isotope-Coded Maleimide Affinity Tags for Proteomics Applications

Abstract

Isotope-coded affinity tags (ICATs) are valuable tools for mass spectrometry-based quantitative proteomics, in particular, for comparison of protein (cysteine-residue) thiol oxidation state in normal, stressed, and diseased tissue. However, the iodoacetamido electrophile used in most commercial ICATs suffers from poor thiolselectivity and modest rates of adduct formation, which can lead to spurious results. Hence, we designed and synthesized three ICATs containing thiol-selective N-alkylmaleimide electrophiles (isotopecoded maleimide affinity tags = ICMATs) and assessed these as mass spectrometry probes for ratiometric analysis of lysozyme and muscle proteomes. Two ICMAT pairs containing butylene/D 8 -butylene linkers were effective MS probes, but not ideal for typical proteomics workflows, because peptides bearing these tags frequently did not coelute with HPLC. A switch to a phenylene/ 13 C 6 -phenylene linker solved this issue without compromising the efficiency of adduct formation.

Enhancing Cysteine Chemoproteomic Coverage through Systematic Assessment of Click Chemistry Product Fragmentation

“…Alongside providing an improved fundamental understanding of how chemoproteomics samples behave in the gas phase, our study offers several added benefits. First, we present the low-cost synthesis of an isotopically labeled pair of biotin-azide reagents, which compares favorably to the cost and complexity of established isotopically labeled reagents, both azide-containing and those that feature cysteine-reactive electrophiles. ,,, Our demonstration of the labile ion search features built into FragPipe should also provide a generalizable computational platform for others interested in leveraging fragmentation of chemoproteomics samples. Exemplifying the utility of such studies, gas-phase fragmentation of cysteines modified by covalent drugs, such as ibrutinib, has been leveraged to improve the identification of labeled cysteine residues. , In addition, fragmentation of sulfonyl-triazole probes has been harnessed for site-of-labeling studies .…”

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology

“…Targeted label-free data-acquisition can be performed with Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) [206,207]. Quantitative label-based methods used in MS-based analyses include stable isotope labelling using amino acids in cell culture (SILAC) [208,209], the isobaric tagging for relative and absolute quantitation (iTRAQ) technique [196,210], isotope-coded affinity tags (ICAT) [211,212], isotope-coded protein labelling (ICPL) [213] and isobaric tandem mass tagging (TMT) [197,214].…”

Maximizing analytical precision: exploring the advantages of ratiometric strategy in fluorescence, Raman, electrochemical, and mass spectrometry detection

“…Various stable isotope labeling methods have been developed for accurate and precise quantification of peptides in mass spectrometry-based proteomics (Figure 2). One such method is Isotope-coded affinity tags (ICAT), which employ a chemical labeling approach to attach a specific tag to the thiol group of cysteine residues in proteins (Wdowiak et al, 2021). By selectively labeling proteins or peptides containing cysteine residues with light or heavy isotopes, MALDI-TOF-MS can be used to generate peptide-related mass spectra for quantificative anaysis.…”