High-throughput identification of in-gel digested proteins by rapid, isocratic HPLC/MS/MS.
The high sensitivity and accuracy of mass spectrometry for identifying proteins has led to an explosive expansion of proteomics research, necessitating rapid procedures for HPLC/MS/MS analysis. Current HPLC/MS/MS analysis usually relies on elution of peptides from the HPLC column with a gradient that takes a total of 45-70 min for each cycle, limiting sample throughput and the speed of protein identification. Here we report a simple method for high-throughput protein identification, using isocratic, either methanol- or acetonitrile-based buffer systems, HPLC elution into an LTQ mass spectrometer. This procedure allows each cycle of highly sensitive HPLC/MS/MS analysis to be completed in 5 min, thus boosting the efficiency of HPLC/MS/MS analysis 9-14-fold. Using this method, each operator can acquire HPLC/MS/MS data for 96 in-gel proteolytic digests in one 8-h working day. The method can easily be implemented in any laboratory with an LTQ mass spectrometer. This protocol should find wide application in mass spectrometry laboratories that require high-throughput analysis but are limited by inefficient use of machine time.
Chen
Yue
Y
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038, USA.
Kim
Sung Chan
SC
Zhao
Yingming
Y
D000097:Acetonitriles / Q000737:chemistry; D001839:Bombesin / Q000378:metabolism; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005136:Eye Proteins / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D010446:Peptide Fragments / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013995:Time
D000097:Acetonitriles; D002021:Buffers; D005136:Eye Proteins; D010446:Peptide Fragments; D011506:Proteins; C052615:peptide M, retinal S antigen; D001839:Bombesin; D000432:Methanol; C032159:acetonitrile