Liquid chromatography MALDI MS/MS for membrane proteome analysis.
Liquid chromatography (LC) can be combined with matrix-assisted laser-desorption ionization (MALDI) mass-spectrometry (MS) by using automated off-line fraction collection of eluates onto a MALDI plate. Commercial tandem MS systems are available for generating product ion spectra of MALDI-produced peptide ions. The MALDI MS/MS spectra can be searched against a proteome database for protein identification. In this chapter, a protocol of sequential solubilization and digestion of membrane proteins involving methanol- and SDS-assisted trypsin digestion and microwave-assisted acid hydrolysis is presented. The process of LC eluate deposition onto a MALDI plate along with practical considerations for achieving optimal performance of eluate deposition is described. Issues related to MALDI MS and MS/MS spectral acquisition are discussed. Database searching and manual inspection of MS/MS spectra of singly charged MALDI-produced peptide ions for positive protein identification are also addressed.
Wang
Nan
N
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Young
J Bryce
JB
Li
Liang
L
D000143:Acids / Q000737:chemistry; D000818:Animals; D002853:Chromatography, Liquid; D030562:Databases, Protein; D006868:Hydrolysis; D008099:Liver / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D008872:Microwaves; D040901:Proteomics / Q000379:methods*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism; D015027:Zebrafish
D000143:Acids; D008565:Membrane Proteins; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin; D000432:Methanol