Comparison between the matrices alpha-cyano-4-hydroxycinnamic acid and 4-chloro-alpha-cyanocinnamic acid for trypsin, chymotrypsin, and pepsin digestions by MALDI-TOF mass spectrometry.
The performance of the recently developed 4-chloro-alpha-cyanocinnamic acid (Cl-CCA) matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) matrix was investigated in comparison to the most widely used matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). For this purpose, in-solution digestions of standard proteins in the low femtomole range with the proteases trypsin, chymotrypsin, and pepsin were used as analytes. For all protein-protease combinations, Cl-CCA revealed to be highly superior in terms of number of identified peptides, obtained sequence coverages and peptide detection reproducibility. A deeper inspection of the detected peptide signals with regard to both physicochemical peptide properties (their isoelectric point) and mass spectrometric performance (signal-to-noise ratios and mass accuracies) showed that the progress achieved with Cl-CCA is due to the detection of numerous acidic to neutral peptides. Moreover, the higher Cl-CCA sensitivity allowed for the detection of numerous additional phosphopeptides, all of which were verified by means of MS/MS investigations. The occurrence of strong signals of doubly charged peptides which is exclusively observed for the Cl-CCA matrix can be traced back to the peptide amino-acid composition, that is, the presence of a high number of basic amino acids (Arg, Lys, and His) and is thus more pronounced for nontryptic protein digests. These observed improvements well agree with an increased protonation reactivity of Cl-CCA and are more pronounced with a decreasing level of protease specificity and decreasing sample amounts.
Cluster of Excellence Macromolecular Complexes, Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt, Germany.
Thorsten W
TW
Jaskolla
Dimitrios G
DG
Papasotiriou
Michael
M
Karas
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry*; D002934:Cinnamates / Q000737:chemistry*; D003373:Coumaric Acids / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D010434:Pepsin A / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*
C532442:4-chloro-alpha-cyanocinnamic acid; D002934:Cinnamates; D003373:Coumaric Acids; D010455:Peptides; D012710:Serum Albumin, Bovine; C007175:alpha-cyano-4-hydroxycinnamate; D002918:Chymotrypsin; D014357:Trypsin; D010434:Pepsin A