An evaluation of MALDI-TOF MS experiments provided first hints towards the

An evaluation of MALDI-TOF MS experiments provided first hints

towards the detectability of methylated peptides via MS

To find out, whether methylated Htr peptides can in principle be detected via MS, MALDI- TOF (matrix-assisted laser desorption/ionization-time of flight) MS spectra, which had led to the identification of Htrs in the course of the ongoing lab-project of H. salinarum proteome analysis (Klein et al., 2005; Tebbe et al., 2005), were checked for the presence of peaks corresponding to putatively methylated peptides from these Htrs. These spectra had been obtained from peptide mixtures, which were generated by tryptic in-gel digest of silver-stained protein spots on 2-D gels of either cytosolic or membrane protein preparations.

Using the computer program Mascot (Matrix Science Inc., Boston, MA, USA), searches had been performed against a database containing all sequences of H. salinarum proteins that were predicted from the complete annotated genome sequence (http://www.halolex.mpg.de). These searches used the peak lists generated via MALDI-TOF MS of the described peptide mixtures. Spectra of those gel spots, in which one or more Htrs had previously (in the course of the proteome analysis) been identified by Mascot via an automated search algorithm called peptide mass fingerprinting (PMF), were checked manually for the presence of peaks, which correspond to the masses of peptides that contain potentially methylatable residues. Such peptides had been determined before as candidates for methylation by inspection of positions 2 and 3 of the relevant heptads, as described above for Htr14. In a number of cases, pairs of peaks with a mass difference of 14 Da were detected, in which the single peaks were consistent with the masses calculated for the unmethylated and the singly methylated form of such peptides. The difference of 14 Da is in accordance with the mass of a methylene group, which comprises the difference between a carboxyl group and its methyl ester. The described peak pairs were detected for some membrane-associated (Htrs 1, 2, 3, 4, and 6) as well as presumably cytosolic Htrs (Htrs 11 and 13). A selection is shown in Fig. 3.22.

For peptide A186 - R208 from cytosolic Htr13 and peptide A513 - R544 from membrane- associated Htr6, the masses observed in the spectra point to a deamidation of the pair's Gln residue to a Glu. The phenomenon of deamidation has been described for E. coli transducers but this was the first indication that deamidation also exists in H. salinarum. The peak observed at the mass corresponding to the deamidated and singly methylated form of the Htr13 peptide A186 - R208 has a much lower intensity than that corresponding to the unmethylated form of the deamidated peptide. This suggests that Htr13 is methylatable at this site (probably only after

preceding deamidation), but in the investigated protein sample only a small fraction of Htr13 was actually carrying a methyl group there.

Fig. 3.22G shows a complete spectrum of a peptide mix, which results from a 2-D gel spot containing Htr3 and Htr6. Three isolated peak pairs, each comprising a difference of 14 Da, can

Figure 3.22 MALDI-TOF mass spectra of some methylatable Htr peptides resulting from tryptic digests of protein spots from 2-D gels.

Proteins were obtained from a total membrane protein preparation of strain TOM (A, B, C, F, G) or from a fraction of a cytosolic preparation of the same strain, generated by size exclusion chromatography (D, E). Samples were prepared and subjected to 2-D gel electrophoresis, tryptic digest and MALDI-TOF MS analysis, as described in Materials and Methods. (A-F) Arrows mark the exact positions on the m/z axis that correspond to the masses of the unmethylated and singly methylated Htr peptides after protonation ([M-H]+_{), as listed above the spectrum. In the}

case of E and F the observed masses are 1 Da higher than calculated but match exactly if a deamidation is assumed that converts the Gln of each peptide to a Glu. (G) Complete spectrum obtained from a gel spot containing Htr3 and Htr6. Peaks matching the calculated masses within a range of < 200 ppm are attributed to peptides from Htr3 (open diamonds) and Htr6 (filled diamonds). Peaks which are frequently observed in other MALDI spectra of similar preparations were marked t (for trypsin fragments) and f (for other unassigned frequently observed peaks). Note the pairs of peaks differing by 14 Da (the mass of a CH2-group) which are centered at approx. 2484, 3158 and 3843 Da.

be clearly seen and correspond to the masses expected for the unmethylated and singly methylated forms of two peptides from Htr3 and one from Htr6.

Although this analysis of MALDI-TOF MS spectra has demonstrated for the first time, that methylated Htr peptides can be detected via MS and that the methylation sites are present in peptides which were predicted to be methylated, the approach has several drawbacks (Table 3.2). (i) Only 11 of the 18 Htrs could be identified as yet via this method. (ii) The putatively methylatable peptides produced by tryptic digest of Htrs are rather large, due to a low content of Lys and Arg residues in the methylatable regions. The peptide sizes therefore often exceed the detection limit of the method, or the detected mass peaks have only a low intensity, hardly above the background. (iii) MALDI-TOF MS analysis cannot unambiguously assign a particular peptide to a mass peak, due to the lack of additional fragmentation information. (iv) Furthermore, no information is provided about the methylation positions within the peptides. Table 3.2. Comparison of the number of residues in those Htr-peptides that contain the putative methylation site at heptad position -12 and were generated by digestion with either trypsin or Asp-N.

Htr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

tryptic peptide a _{30 40 38 33 39 32 42 28 59 20 31 49 23 48 84 56 35 33}

Asp-N- peptide b _{16 17 17 14 13 22 18 13 32 20 28 13 17 15 31 21 21 13}

a: Underlining indicates that peaks with masses consistent with those of the unmethylated and singly methylated forms of the corresponding peptide were identified via MALDI-TOF MS in the course of the H. salinarum

proteome analysis project in the lab. Italicized numbers correspond to peptides which exceed the detection range of the MS setup (800 - 4,000 Da). Strike-through indicates that the corresponding Htrs have, so far (until August 2005), completely escaped an identification via MALDI-TOF MS.

b: Double underlining indicates that the corresponding peptide was identified via LC ESI Q-TOF MS/MS in the course of this study.