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A rapid and easy protein N-terminal profiling strategy using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) labeling and StageTip
- Source :
- Proteomics. 17(13-14)
- Publication Year :
- 2016
-
Abstract
- Protein N-terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N-terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N-terminal peptide losses. In this study, a fast, one-step approach utilizing (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N-terminal peptide enrichment and analysis. Based on the characteristics of TMPP-derivatized samples, such as a higher hydrophobicity and increased likelihood to produce a and b ions in collision-induced dissociation or HCD fragmentation modes, first the SDS-PAGE was optimized to increase protein loading and gel entry and to remove unbound TMPP. Then, this process was combined with a simplified StageTip separation and a new scoring criterion (considering a, b and y ions) to identify more TMPP-modified N-terminal spectra. When utilizing a low amount of starting material (∼20 μg protein), a total of 581 yeast N-terminal peptides were identified, with 485 of them being TMPP modified, in only about one third of the general experimental time. It is hoped that the workflow constructed herein will provide a fast and practical strategy for N-terminomic studies.
- Subjects :
- 0301 basic medicine
Tris
Proteomics
Saccharomyces cerevisiae Proteins
Peptide
Saccharomyces cerevisiae
Chemical Fractionation
Biochemistry
03 medical and health sciences
chemistry.chemical_compound
Onium Compounds
Organophosphorus Compounds
Protein Domains
Bromide
Phosphonium
Derivatization
Molecular Biology
Cells, Cultured
chemistry.chemical_classification
Chromatography, Reverse-Phase
Chromatography
Chemistry
Hydrogen-Ion Concentration
Peptide Fragments
Chromatographic separation
030104 developmental biology
Protein loading
Subjects
Details
- ISSN :
- 16159861
- Volume :
- 17
- Issue :
- 13-14
- Database :
- OpenAIRE
- Journal :
- Proteomics
- Accession number :
- edsair.doi.dedup.....f1c922258a0e5268a902c03233e719dd