S-pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: A rapid mass spectrometric method for identifying cysteine-containing peptides

被引:88
作者
Moritz, RL [1 ]
Eddes, JS [1 ]
Reid, GE [1 ]
Simpson, RJ [1 ]
机构
[1] ROYAL MELBOURNE HOSP, WALTER & ELIZA HALL INST MED RES, JOINT PROT STRUCT LAB, PARKVILLE, VIC 3050, AUSTRALIA
关键词
two-dimensional gel polyacrylamide gel electrophoresis; mass spectrometry; peptide mass fingerprinting; capillary liquid chromatography;
D O I
10.1002/elps.1150170512
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
In-gel proteolytic digestion of acrylamide-gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide-mass fingerprinting. However, it is well recognised for disulfide-bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result. Here, we describe an improved method for in-gel protein digestion. It consists of first reducing and S-pyridylethylating Coomassie Brilliant Blue R-250-stained proteins immobilised in the whole gel slab with dithiothreitol and 4-vinylpyridine, excising the individual stained and alkylated proteins, and then digesting them in situ in the gel matrix with trypsin or Achromobacter lyticus protease I. Peptide fragments generated in this manner are extracted from the gel piece and purified to homogeneity by a rapid (less than or equal to 12 min) reversed-phase high performance liquid chromatography (HPLC) procedure, based upon conventional silica supports. Recoveries of peptides are increased by S-pyridylethylation of acrylamide-immobilised proteins prior to in-gel digestion. Further, the levels of gel-related contaminants, which otherwise result in suppression of sample signals during electrospray-ionisation mass spectrometry, are greatly reduced by the reduction / alkylation step. Additionally, we demonstrate that S-beta-(4-pyridylethyl)-cysteine containing peptides can be readily identified during reversed-phase HPLC by absorbance at 254 nm, and during electrospray ionisation tandem mass spectrometry by the appearance of a characteristic-pyridylethyl fragment ion of 106 Da. The position of cysteine residues in a sequence can be determined as phenylthiohydantoin S-beta-(4-pyridylethyl)-cysteine during Edman degradation, and by tandem mass spectrometry.
引用
收藏
页码:907 / 917
页数:11
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