Fragmentation reactions of protonated α-amino acids (AAs) were studied previously using tandem mass spectrometry (MS/MS) of unit mass resolution. Isobaric fragmentation products and minor fragmentation products could have been overlooked or misannotated. In the present study, we examined the fragmentation patterns of 19 AAs using high-resolution electrospray ionization MS/MS (HR-ESI-MS/MS) with collision-induced dissociation (CID). Isobaric fragmentation products from protonated Met and Trp were resolved and identified for the first time. Previously unreported fragmentation products from protonated Met, Cys, Gln, Arg, and Lys were observed. Additionally, the chemical identity of a fragmentation product from protonated Trp that was incorrectly annotated in previous investigations was corrected. All previously unreported fragmentation products and reactions were verified by pseudo MS 3 experiments and/or MS/MS analyses of deuterated AAs. Clearer pictures of the fragmentation reactions for Met, Cys, Trp, Gln, Arg and Lys were obtained in the present study.
Covalent inhibitors with an electrophilic warhead have received considerable attention due to their remarkable pharmacological properties. However, the electrophilic warhead in covalent drugs is often an α, β-unsaturated amide, and the targets are mainly cysteine or lysine residues. Thus, the development of novel electrophiles that can target other amino acids is highly desirable. Ynamide, a useful and versatile building block, is commonly employed in the construction of various compounds in organic synthesis. The performance of this functional group in a proteome-wide environment has been studied here for the first time, and it has been shown that it can efficiently modify carboxyl residues in situ and in vitro. Upon incorporation of this ynamide warhead into the pharmacophores of kinase inhibitors, the resulting compound showed moderate inhibition against the EGFR L858R mutant but not against EGFR WT. This novel electrophilic group can be used in the development of new types of covalent inhibitors.
Cystine is an important biomolecule in living systems. Although collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) is commonly applied for identification and quantification of cystine in both biomedical and nutritional studies, gas-phase fragmentation reactions of cystine in CID has remained unclear. This may lead to improper assay design, which may in turn result in inaccurate test results. In the present study, gas-phase fragmentation reactions of protonated cystine in CID were characterized using high-resolution MS/MS and pseudo MS3. Fragmentations started from cleavages of disulfide bond (S–S) and carbon–sulfur bond (C–S). When cleaving at the S–S, protonated cysteine was generated as one of the predominant fragmentation products. Minor fragmentations started from the loss of H2O + CO and the loss of NH3. Our results reveal that the m/z 74 fragment ion, which is commonly used as a product ion of the transition (precursor/product ion pair) in selected reaction monitoring (SRM) assay for quantifying cystine, comprises two isobaric fragments originating from different parts of cystine. This indicates the need for careful selection of a stable isotope-labeled cystine molecule as an internal standard for SRM assays. Here, we provide a clear picture of the fragmentation reactions of protonated cystine in CID. It can serve as a useful guidance for designing MS/MS-based assays for cystine testing.
Dear Editor, Our study demonstrates that biomarker research using liquid chromatography (LC)-high resolution (HR) mass spectrometry (MS) based untargeted metabolomics profiling is susceptible to the discovery of false positive biomarkers.LC-MS, especially LC-HRMS, is popularly used to discover putative biomarkers through comparing untargeted metabolomic profiles between a patient group and a control group. 1,2 This approach is susceptible to various preanalytical, analytical, and post-analytical biases. 3 Moreover, isotopes, adducts, in-source fragment products of some metabolites, artifacts, and contaminants could be wrongly considered as unique metabolomic features. 4,5 To what extent the putative metabolomic biomarkers could be false remains unknown.We attempted to identify putative biomarkers for differentiating two artificial groups of plasma samples (12 samples in each group) with well-defined differences in their metabolome contents (Figure 1A, Table 1; Tables S1 and S2). By design, a maximum of 22 putative biomarkers are true, and the rest of the putative biomarkers must be false.The number of metabolomic features depended on the signal-to-noise ratio threshold (snthresh) used for feature extraction (Table 2). A snthresh of 5 had been widely used (Table S3). Using a snthresh of 5 and a false discovery rate (FDR) cutoff of 5% for data mining, 22 true biomarkers (i.e., true positives) and 165 false positive biomarkers were observed (Table 3; Table S4). Therefore, the actual FDR (i.e., the false positive rate) was 88% instead of 5% (Table 2). Increasing the snthresh and reducing the sample size (e.g., n = 6 for each group) could decrease the number of false positive biomarkers. However, the actual FDR remained > 60% (Table 2; Table S5). We also performed a negative control experiment using two groups of plasma samples (n = 12 for each group) having identical metabolome contents. No differential metabolomic features were found (Supplemental Table S6). This indi-This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Emerging evidence suggests that advanced glycation end-products (AGEs) such as N ε -(carboxymethyl)lysine (CML) and N ε -(carboxymethyl)lysine (CEL) may play important roles in certain human diseases. Reliable analytical methods are needed for their characterizations and measurements. Pitfalls have been reported for applications of LC−MS/MS to identify various types of post-translational modifications, but not yet for the case of AGEs. Here, we showed that in the absence of manual inspection, cysteine alkylation with 2-iodoacetamide (IAA) can result in false-positive/ambiguous identifications of CML >20%. They were attributed to offsite alkylation together with incorrect monoisotopic peak assignment (pitfall 1) or together with deamidation (pitfall 2). For pitfall 1, false-positive identifications can be alleviated using a peptide mass error tolerance ≤5 ppm during the database search. Pitfall 2 results in ambiguous modification assignments, which may be overcome by using other alkylation reagents. According to calculations of theoretical mass shifts, the use of other common alkylation reagents (iodoacetic acid, 2-chloroacetamide, and acrylamide) should face similar pitfalls. The use of acrylamide can result in false-positive identifications of CEL instead of CML. Subsequently, we showed that compared to IAA, the use of N-isopropylacrylamide (NIPAM) as an alkylation reagent achieved similar levels of proteome coverage, while reducing the offsite alkylation reactions at lysine by more than five times. Furthermore, false-positive/ambiguous identifications of CML due to the two types of pitfalls were absent when using NIPAM. NIPAM alkylation results in a unique mass shift that allows reliable identifications of CML and most likely other AGEs, such as CEL.
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