The analysis of myocardial proteins by infrared and ultraviolet laser desorption mass spectrometry.
The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.
Sutton
C W
CW
Thermo BioAnalysis Ltd., Hemel Hempstead, UK.
Wheeler
C H
CH
Sally
U
U
Corbett
J M
JM
Cottrell
J S
JS
Dunn
M J
MJ
D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000378:metabolism; D006352:Heart Ventricles / Q000737:chemistry; D006801:Humans; D007259:Infrared Rays; D008970:Molecular Weight; D009206:Myocardium / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays
D011506:Proteins; D010450:Endopeptidases