Peptide mass fingerprinting after less specific in-gel proteolysis using MALDI-LTQ-Orbitrap and 4-chloro-alpha-cyanocinnamic acid.
Peptide Mass Fingerprinting (PMF) of tryptically in-gel digested samples is a well-established protein identification technique for MALDI mass spectrometry but an in-depth PMF evaluation for in-gel digestions of less specific enzymes is still missing. This study demonstrates that the MALDI-LTQ-Orbitrap provides the mass accuracy to gain significant database search results via PMF for the less specific enzymes chymotrypsin and elastase. Additionally, the highly sensitive MALDI matrix ClCCA was compared to the most widely used matrix CHCA by means of the detected peptide number, peptide composition, pI and S/N distribution, sequence coverage, and Mascot score. Therefore, several proteins were in-gel digested by chymotrypsin and elastase. Trypsin and proteinase K were included as references for specific and nonspecific proteases, respectively. Compared to CHCA, ClCCA resulted in a better mapping in all cases of the more complex peptide mixtures generated by less specific enzymes. In summary, the MALDI-LTQ-Orbitrap combined with the matrix ClCCA makes PMF of less specific digests possible in an easy and fast way. Moreover, it opens more possibilities for PMF in the analysis of difficult tasks such as membrane proteins.
Papasotiriou
Dimitrios G
DG
Cluster of Excellence Macromolecular Complexes, Institute of Pharmaceutical Chemistry, Goethe-University, D-60438 Frankfurt am Main, Germany.
Jaskolla
Thorsten W
TW
Markoutsa
Stavroula
S
Baeumlisberger
Dominic
D
Karas
Michael
M
Meyer
Bjoern
B
D002918:Chymotrypsin / Q000378:metabolism*; D002934:Cinnamates / Q000737:chemistry* / Q000378:metabolism; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D010196:Pancreatic Elastase / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
C532442:4-chloro-alpha-cyanocinnamic acid; D002934:Cinnamates; D003373:Coumaric Acids; D010446:Peptide Fragments; D011506:Proteins; C007175:alpha-cyano-4-hydroxycinnamate; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin