pChem: a modification-centric assessment tool for the performance of chemoproteomic probes

He, Ji-Xiang; Fei, Zhengcong; Fu, Ling; Tian, Caiping; He, Fuchu; Chi, Hao; Yang, Jing

doi:10.1101/2021.09.22.461295

Cited by 4 publications

(5 citation statements)

References 87 publications

(85 reference statements)

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“…The last decade has witnessed tremendous progress in the development of blind search tools that can provide an opportunity to explore unexpected modifications formed in the proteome. − Hence, we sought to use one of such tools called pChem to provide an unbiased survey of all possible modifications derived from the tested tags (Figure S9). Surprisingly, the pChem search identified, in addition to the known Mod 3 (Δ238.14 Da) derived from 3 (DADPS), a mass shift of 266.14 Da that occurs dominantly on cysteine (i.e., the probe-targeting residue, Figure A) while revealing the uniformity of modifications derived from other cleavable biotin tags.…”

Section: Resultsmentioning

confidence: 99%

“…Raw files were searched against the defaulted SwissProt Homo sapiens database downloaded from Uniprot on May 16, 2021 (20,395 entries) using pChem (version 1.0) (). Common modifications (i.e., oxidation of methionines and carbamidomethylation of cysteines) were predefined to improve search sensitivity.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, the software calculated Ascore for each cysteine site in order to evaluate the confidence of site localization. 17 Raw files were searched against the defaulted SwissProt Homo sapiens database downloaded from Uniprot on May 16, 2021 (20,395 entries) using pChem (version 1.0) 18 (http://pfind. org/software/pChem/index.html).…”

Section: Lc−ms/msmentioning

confidence: 99%

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Benchmarking Cleavable Biotin Tags for Peptide-Centric Chemoproteomics

Liu

et al. 2022

J. Proteome Res.

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Click chemistryspecifically the copper-catalyzed azide-alkyne cycloadditionhas enabled the development of a wide range of chemical probes to analyze subsets of the functional proteome. The "clickable" proteome can be selectively enriched by using diverse cleavable biotin tags, but the direct identification of probe/tag-modified peptides (or peptide-centric analysis) remains challenging. Here, we evaluated the performance of five commercially available cleavable biotin tags in three most common chemoproteomic workflows, resulting in a comparative analysis of 15 methods. An acidcleavable biotin tag with a dialkoxydiphenylsilane moiety, in combination with the protein "click", peptide "capture" workflow, outperforms all other methods in terms of enrichment efficiency, identification yield, and reproducibility, although its performance may be slightly compromised by the formation of an unwanted formate product revealed by blind search. Despite being inferior, photocleavable, and reduction-cleavable, biotin tags can also find their applicable sceneries, especially when dealing with acid-sensitive probes or probe-derived modifications. Furthermore, the systematic comparison of LC−MS/MS characteristics of tag-modified peptides provides valuable information for the future development of cleavable biotin reagents. Taken together, our data provides a much-needed practical guidance for researchers interested in developing and/or applying an ideal cleavable biotin tag to peptide-centric chemoproteomics.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Benchmarking Cleavable Biotin Tags for Peptide-Centric Chemoproteomics

Liu

et al. 2022

J. Proteome Res.

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“…Chemoproteomics has a particular stake in this effort due to the diversity of probes employed [17][18][19] . However, existing tools for chemoproteomics require isotopic labeling signatures to be present at the MS1 and MS2 levels 20 . This limits their applications to chemical probes that are labeled nonisobarically, thus they cannot be used for some PTM probes 21 , biological PTMs, or the development of isobaric mass tags 22 .…”

Section: Introductionmentioning

confidence: 99%

Mining for ions: diagnostic feature detection in MS/MS spectra of post-translationally modified peptides

Geiszler

Polasky

et al. 2022

Preprint

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Post-translational modifications (PTMs) are an area of great interest in proteomics, with a surge in methods to detect them in recent years. However, PTMs can introduce complexity into proteomics searches by fragmenting in unexpected ways. Detecting post-translational modifications in mass spectrometry-based proteomics traditionally relies on identifying ions shifted by the masses of the modifications. This presents challenges for many PTMs. Labile PTMs lose part of their modification mass during fragmentation, rendering shifted fragment ions unidentifiable, and isobaric PTMs are indistinguishable by mass, requiring other diagnostic ions for disambiguation. Furthermore, even modifications that have undergone extensive characterization often produce different fragmentation patterns across instruments and conditions. To address these deficiencies and facilitate the next generation of PTM identification, we have developed a method to automatically find diagnostic spectral features for any PTM, allowing subsequent searches to take advantage of additional metrics and increase PTM identification and localization rates. The method has been incorporated into the open-search annotation tool PTM-Shepherd and the FragPipe computational platform.

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“…, modifications) derived from a chemical probe and to unbiasedly assess its proteome-wide residue selectivity. 38,39 We therefore sought to use one of such tools termed pChem 38 to re-analyse the MS data (see Methods, ESI † ). Surprisingly, the pChem search identified three new and abundant PDMs ( Fig.…”

mentioning

confidence: 99%