Screening for transglutaminase-catalyzed modifications by peptide mass finger printing using multipoint recalibration on recognized peaks for high mass accuracy

Abstract

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.

FIGURE 1

MALDI mass spectrum used to screen for TG-susceptible Lys and Gln residues. Mass spectrum showing a tryptic digest of human Hsp27 treated by TG in the presence of the hexapeptide probe GQDPVR for Lys labeling. After export of peak mass values for spectrum analysis, multipoint recalibration on recognized peaks was performed using the programs MoverZ, GPMAW, and PeakErazor as outlined in the protocol in Scheme 1 and Figures 2 and 3. After such spectrum analysis, two peaks were identified as peptides containing the C-terminal Lys, K205, modified by formation of a covalent isopeptide bond with the hexapeptide probe GQD-PVR (arrows). That the C-terminal Lys is susceptible to TG was previously documented using a similar hexa-peptide probe in combination with exoprotease treatment, both for αB-crystallin and for Hsp27.

FIGURE 2

Multipoint recalibration on recognized peaks. Screen-shots of the program PeakErazor used for multipoint recalibration on recognized peaks as outlined in the protocol in Scheme 1. Peak mass value lists of peptides observed are imported from mass spectra into PeakErazor, and matched against peak lists derived from theoretical digests in silico of the sequence from the analyzed protein, annotated as , and an Erazor list, intrinsic to PeakErazor, derived from theoretical digests in silico of the sequences for keratin and trypsin. Screen-shots illustrate appearance before (A) and after (B) the linear multipoint calibration, which is based on these matched and recognized peaks. Values presented in this figure are for Hsp20 (P97541 in Swiss-Prot).

FIGURE 2

Multipoint recalibration on recognized peaks. Screen-shots of the program PeakErazor used for multipoint recalibration on recognized peaks as outlined in the protocol in Scheme 1. Peak mass value lists of peptides observed are imported from mass spectra into PeakErazor, and matched against peak lists derived from theoretical digests in silico of the sequence from the analyzed protein, annotated as , and an Erazor list, intrinsic to PeakErazor, derived from theoretical digests in silico of the sequences for keratin and trypsin. Screen-shots illustrate appearance before (A) and after (B) the linear multipoint calibration, which is based on these matched and recognized peaks. Values presented in this figure are for Hsp20 (P97541 in Swiss-Prot).

FIGURE 3

Searching for modified peaks using GPMAW with user-defined modifications. Searching for modified peaks is performed in the program GPMAW using user-defined modifications as outlined in the protocol in Scheme 1. Screen-shot from the program GPMAW showing the report from a mass search in the Hsp20 sequence (P97541 in Swiss-Prot), with user-defined modifications for Hsp20 incubated with TG and the hexapeptide probe GNDPVK. Upper part shows a graphic presentation of the sequence coverage (in this case 46%). Lower part shows the number of matched peptides (in this case 5) with mass deviations, 10 ppm, and the number of matched peptides with modifications (in this case 2), one peptide (aa 28–32) containing Hsp20 Q31 modified by Gln cross-linking to the hexapeptide probe GNDPVK, and one peptide (aa 57–81) containing Hsp20 Q66 with Gln deamidation to Hsp20 E66. Further below unmatched peptides are reported, and at the bottom also a list of all the user-defined modifications used in the GPMAW mass search.

FIGURE 4

Hexapeptide probe cross-linking and appearance of modified peaks at the expense of unmodified peaks in Hsp20. Overview of MALDI mass spectra recorded for Hsp20 incubated with TG in presence of the hexapeptide probe GQDPVR for labeling of Lys residues (upper), or the hexapeptide probe GNDPVK for labeling of Gln residues (middle), and control Hsp20 without TG (lower). Mass spectra are magnified to show different mass ranges, m/z below 1700 Da (A) or m/z above 3800 Da (B). Filled and unfilled arrows mark the peptides, which increase and decrease in intensity, respectively.

FIGURE 4

Hexapeptide probe cross-linking and appearance of modified peaks at the expense of unmodified peaks in Hsp20. Overview of MALDI mass spectra recorded for Hsp20 incubated with TG in presence of the hexapeptide probe GQDPVR for labeling of Lys residues (upper), or the hexapeptide probe GNDPVK for labeling of Gln residues (middle), and control Hsp20 without TG (lower). Mass spectra are magnified to show different mass ranges, m/z below 1700 Da (A) or m/z above 3800 Da (B). Filled and unfilled arrows mark the peptides, which increase and decrease in intensity, respectively.

FIGURE 5

Cross-linking by intramolecular peptide bond formation in Hsp20. MALDI mass spectra recorded for Hsp20 incubated with TG (A) and control Hsp20 without TG (B). A band, detected below the hexapeptide probe labeled Hsp20 (see Fig. 1, Hsp20 in Boros et al., reference 23), was investigated for a presumed intramolecular cross-link. A cross-linked peptide representing an intramolecular Q31-K162 isopeptide bond formation was detected as two peaks (marked by green arrows) appearing at 3763.0 Da (LFDQ31R covalently cross-linked to LPP. . .PAAK162) and 4423.32 Da (LFDQ31R covalently cross-linked to LPP. . .PAAK162), respectively. The peaks representing corresponding unmodified peptides decreased correspondingly (marked by white arrows ).