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High‐efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry
Author(s) -
Jin Ya,
Manabe Takashi
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200410187
Subject(s) - mass spectrometry , chromatography , chemistry , molecular mass , coomassie brilliant blue , matrix assisted laser desorption/ionization , extraction (chemistry) , time of flight mass spectrometry , polyacrylamide gel electrophoresis , protein mass spectrometry , protein purification , matrix (chemical analysis) , sample preparation in mass spectrometry , desorption , biochemistry , tandem mass spectrometry , ionization , electrospray ionization , enzyme , staining , ion , biology , organic chemistry , adsorption , genetics
A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)‐stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) is reported. Proteins in CBB‐stained gel pieces were extracted by a 10‐min soaking in 0.1 M NaOH at 25°C. The recovery of this one‐step extraction method was 34–73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within ±0.01–0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB‐stained gels with loaded protein quantities as little as 34 ng for cytochrome c , α‐lactalbumin, myoglobin, β‐lactoglobulin, trypsinogen, and carbonic anhydrase (12.4–29.0 kDa), 340 ng for glyceraldehyde‐3‐phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB‐stained one‐ or two‐dimensional gels for whole protein analysis using MALDI‐TOF‐MS.