Screening for transglutaminase-catalyzed modifications by peptide mass finger printing using multipoint recalibration on recognized peaks for high mass accuracy.
Detection of posttranslational modifications is expected to be one of the major future experimental challenges for proteomics. We describe herein a mass spectrometric procedure to screen for protein modifications by peptide mass fingerprinting that is based on post-data acquisition improvement of the mass accuracy by exporting the peptide mass values into analytical software for multipoint recalibration on recognized peaks. Subsequently, the calibrated peak mass data set is used in searching for modified peptides, i.e., peptides possessing specific mass deviations. In order to identify the location of Lys- and Gln-residues available for transglutaminase-catalyzed isopeptide bond formation, mammalian small heat shock proteins (sHsps) were screened for labeling with the two hexapeptide probes GQDPVR and GNDPVK in presence of transglutaminase. Peptide modification due to cross-linking of the GQDPVR hexa-peptide probe was detected for C-terminal Lys residues. Novel transglutaminase-susceptible Gln sites were identified in two sHsps (Q31/Q27 in Hsp20 and HspB2, respectively), by cross-linking of the GNDPVK hexapeptide probe. Deamidation of specific Gln residues was also detected, as well an isopeptide derived from intramolecular Gln-Lys isopeptide bond formation. We conclude that peptide mass fingerprinting can be an efficient way of screening for various posttranslational modifications. Basically any instrumentation for MALDI mass spectrometry can be used, provided that post-data acquisition recalibration is applied.
Emanuelsson
Cecilia Sundby
CS
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden. Cecilia.Emanuelsson@biokem.lu.se
Boros
Sandor
S
Hjernoe
Karin
K
Boelens
Wilbert C
WC
Hojrup
Peter
P
D000595:Amino Acid Sequence; D002138:Calibration; D002384:Catalysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000592:standards*; D011503:Transglutaminases / Q000378:metabolism*
D010455:Peptides; D011503:Transglutaminases