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Intensive Protein Digestion Using Cross‐Linked Trypsin Aggregates in Proteomics Analysis
Author(s) -
Wang MengFan,
Mu YangYang,
Qi Wei,
Su RongXin,
He ZhiMin
Publication year - 2013
Publication title -
chempluschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.801
H-Index - 61
ISSN - 2192-6506
DOI - 10.1002/cplu.201200323
Subject(s) - trypsin , chemistry , digestion (alchemy) , proteomics , autolysis (biology) , chromatography , mass spectrometry , lysozyme , denaturation (fissile materials) , bottom up proteomics , bovine serum albumin , biochemistry , peptide , enzyme , protein mass spectrometry , electrospray ionization , nuclear chemistry , gene
In this study, cross‐linked enzyme aggregates of tissue‐culture‐grade trypsin (CLEAs‐T) were prepared and introduced to the proteomics analysis instead of the expensive proteomics‐grade trypsin, which benefits from the protein purification during the preparation of the CLEAs. Bovine serum albumin, lysozyme, bovine hemoglobin, and α‐casein were used as model proteins, and three intensive strategies (high‐enzyme‐concentration, high‐temperature, and ultrasound‐assisted digestion) were applied to the rapid and efficient digestion of model proteins by CLEAs‐T. The peptide fragments were then identified by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, which revealed the higher sequence coverage and sharply reduced digestion time of these strategies compared with the conventional in‐solution 12 h digestion method. Morphology studies demonstrated that the excellent performance of CLEAs‐T in proteomics analysis came from the “scaffoldlike” supermolecular structure, which provided a high resistance to the autolysis and denaturation of trypsin under high‐enzyme‐concentration, high‐temperature, and ultrasound‐assisted digestion.