Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.
In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.
Lamer
S
S
Max-Planck-Institute for Infection Biology, Central Support Unit Biochemistry, Berlin, Germany.
Jungblut
P R
PR
D001426:Bacterial Proteins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D008970:Molecular Weight; D009163:Mycobacterium bovis / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001426:Bacterial Proteins; D010455:Peptides; D020543:Proteome