PTMs found in bovine MBP and those found in other species
Generally, the sequence coverage generated from trypsin digestion was more than 80% and it was more than 60% from Glu-C digestion for a total coverage of more than 90% for the combined methods. A typical false discovery rate for tryptic peptides at the identity threshold was around 1.5% and it was under 3% for those peptides cleaved from Glu-C digestion. At least three variants of bovine MBP appeared to be present in the preparation by SDS gel electrophoresis, a major form corresponding to approximately 18 kDa and minor forms corresponding to approximately 16 kDa, 23 kDa, and 37 kDa. Some of these minor forms were verified by MALDI peptide mass fingerprinting of the gel bands analysis of tryptic digests and altogether were estimated to correspond to less than 2% of the total MBP and thus did not interefere with the intact mass analyses. Rattlesnake MBP was estimated to be 19 kDa by SDS gel electrophoresis.
We identified a total of 12 PTMs in all 5 charge components from bovine MBP: C1–3, C8a and C8b based on LC-MSMS of proteolytic digests (, ). The PTMs identified are: acetylated Ala at the protein N-terminus; oxidized M19; deimination at R23, R47, R96 and R161; deamidation at Q102 and Q120; phosphorylated T97; mono- and dimethylated R106; and acetylated K121.
All PTMs found in this study indicated in the bovine MBP primary sequence
Peptides with PTMs found in Bovine MBP isomers
Among these modifications of bovine MBP, the putative presence of acetylation of a lysyl residue has not been previously reported. The modified peptide identified in this study was consistent with the presence of acetyl-lysine () at position 121, adjacent to a deamidated glutamine at 120. The total mass shift of 43 Daltons is localized to the QKP tripeptide based on the observed fragment ions and was initially interpreted as a potential modification of the lysyl residue in the deamidated peptide. The observed mass (1842.8370 Da) at 2.4 ppm is consistent with the calculated mass for the acetyl-lysine peptide (1842.8326 Da) rather than trimethylysine (1842.7966 Da) at 22 ppm but indistinguishable from the mass of the carbamylated and non-deamidated peptide (1842.8438) at 3.7 ppm. The average experimental mass accuracy for peptides identified in this study is 5 ppm.
MS/MS Fragmentation of 113-FSWGAEGQKPGFGYGGR-129
Inspection of the MSMS spectrum identified a weak fragment ion at 901.18 Da which might correspond to the neutral loss reported for carbamylated peptides24,25
, but the intensity is much lower than expected and the mass assignment is limited by mass accuracy in the MSMS mode. To distinguish between the presence of acetylation or carbamylation on Lys 121, a Western blot of bovine MBP () using anti-acetyllysine antibody was performed. The immunoblot is consistent with the presence of acetyllysine in MBP, with the highest level in component C3. The staining intensity observed for acetyl-lysine immunoreactivity in MBP is consistent with the low spectral count obtained for the putative acetylated form of this peptide (5 spectra were detected in C3, none were found in other components). This modified lysine appears to be a rare modification based on spectral counts of the modified versus non-modified peptide. Note that the observation of a probable acetyl-lysine only in the deamidated peptide may simply be a sampling issue and we cannot exclude the possibility that the parent peptide is not also acetylated.
Confirmation of the acetylated-Lysine in MBP fraction C3 by Western Blot
We identified both mono- and dimethylated forms of R106 in all five MBP components () which verified a previous study1
. The strong neutral losses of 31 Da and 49 Da observed in the MSMS spectra correspond respectively to monomethylamine (MMA, H2N-CH3) and MMA+H2O26
, confirming that R106 was symmetrically dimethylated (). This is consistent with previous reports for other species which used non-mass spectrometric methods1
. The level of methylation observed varied in the different charge components. By intact mass analysis, the ratio of monomethylation vs dimethylation decreased in components C1 through C3 () but it was not possible to verify this in components C8a and C8b because intact mass analysis failed to produce high-quality spectra due to impurities present in these two components. Brostoff, S. et al
., postulated that the biological function of the methylated arginine residue was to stabilize the double-chain structure for the protein induced by the triproline site1
. In human MBP, more highly methylated Arg was observed in multiple sclerosis patients compared with normal tissue and has been suggested to play an important role in the pathogenesis of multiple sclerosis8
. It is noteworthy that this modification has been found in many species such as human, bovine, rabbit, and chicken but was not found in spiny dogfish. We also found clear evidence for this modification in rattlesnake based on intact mass analysis of rattlesnake MBP (). Spectra from MSMS also confirmed this modification in a peptide with the sequence GRGLSFSR () as this sequence matched well with known species and the fragmentation pattern of this peptide is very similar to that of the methylated peptide from bovine MBP (). Absence of this modification in some lower animals such as dogfish may be due to lack of an arginine in this position (), suggesting methylated arginine is widely distributed from mammals to reptiles but absent in MBP from lower vertebrates which lack this residue.
Identification of arginine methylation at position 106 in all five components
Intact mass analysis of 3 bovine MBP components and unfractionated rattlesnake MBP
Identification of arginine methylation in rattlesnake MBP
Comparison of MBP PTMs in different species
Several sites were observed in bovine MBP for conversion of arginine to citrulline. Catalysed by peptidylarginine deiminases (PADs), deimination (citrullination) of arginine residues can have significant consequences for the structure and function of proteins, and has also been implicated in the pathogenesis of multiple sclerosis27–29
. Native MBP contains several non-deiminated arginines and forms tighter, more compact myelin sheaths. Deimination of MBP affects the stability of the myelin sheath by conversion of positively charged arginine into uncharged citrulline, which increases the hydrophobicity of the protein and reduces the interaction with the negatively charged phosphatidylserine in the membrane, leading to a more open structure susceptible to proteolysis by cathepsin D29,30
. The ratio of deiminated MBP/total MBP was found to be crucial in the physiological function of CNS and the degree of deimination of MBP correlated well with the severity of multiple sclerosis28,29
. MBP was highly deiminated in multiple sclerosis patients and in infants during normal CNS development and was less deiminated in healthy adults8,28,29
. More interestingly, possible crosstalk between deimination and methylation in MBP has been revealed by Pritzker et al
who observed that methylation of R106 in bovine MBP (R107 in human MBP) had a significant correlation to arginine deimination. In human MBP, increased deimination of arginyl residues accompanied with a decreased methylation of R107 has been observed. In this study, we found a total of 4 arginines deiminated at positions 23, 47, 96, and 161 respectively (Figure S1–4
). Although deimination of arginine in MBP has been found in other species, deimination has not previously been reported in bovine MBP. The residue corresponding to R23 in bovine MBP has been reported to be deiminated in human and chicken MBP6,8
and it was also observed in rattlesnake MBP in this study (Figure S5
) indicating that deimination is highly conserved at this site. The residues corresponding to R47 and R161 in bovine MBP have only been previously observed to be deiminated in human MBP, while deiminated R96 found in this study has not been found in other species thus far. The conserved Arg in rattlesnake corresponding to bovine R161 was not found to be deiminated but the nearby R (in peptide sequence GYR
YDGQGTLSK, Figure S6
) was found to be partially deiminated (both forms observed by MS). There is no homologous Arg residue in mammalian MBPs. The degree of conservation of deimination in mammalian MBPs is still incomplete as deimination of Arg has not yet been examined in rabbit or other mammalian MBPs.
Acetylated alanine at the protein N-terminus was found in components C1, C2, and C3 in this study. This modification was not found in the previous study of bovine MBP PTMs by capillary electrophoresis-mass spectroscopy2
. And it was also not found in the tryptic peptides in this study possibly because the peptide with N-acetylalanine (AAQK) was too short to be effectively detected by the mass spectrometry method or the peptide did not ionize well. When we digested MBP with Glu-C protease, the N-terminally acetylated peptide was clearly detected and the PTM was unambiguously assigned by tandem mass spectrometry (Figure S7
). Intact protein mass analysis also agreed with our Glu-C bottom-up study (). Bovine MBP in C1–3 appears to be predominantly acetylated since no spectra corresponding to the unmodified peptide or protein were observed in both bottom-up and intact mass analyses. Acetylation of the N-terminal alpha-amino group of proteins is a common modification in eukaryotes and about 85% of eukaryotic proteins are N-terminally acetylated32
, with N-terminal Ala being a common acetylation site. As a conserved and widespread modification, the biological role of this N-terminal acetylation is unclear, although it has been suggested that one biological function is as a signal for protein degradation33
. This modification was also found in other MBPs from mammals such as human, bovine, and rabbit, to non-mammals including chicken, dogfish, and rattlesnake in this study (Figure S8
A wide range of MBP phosphorylation sites have been reported in previous studies1–9
, however, this study did not focus on phosphorylation so no effort was made to enrich for phosphopeptides. Despite the absence of enrichment methods, we identified a phosphorylation site at T97 which was present in bovine MBP components C2 and C3 (Figure S9
Phosphorylation of T97 has been previously reported for human, bovine, rabbit, and chicken MBPs but not in spiny dogfish because the sequence (PRTPPP) associated with this PTM is highly conserved in higher animals but is missing in dogfish. Other conserved phosphorylated sites are: S7 found in human, bovine, rabbit and chicken; Phosphorylated Ser in sequence RGSGK corresponding to S54 in bovine, and S56 in human and rabbit; Phosphorylated Ser at protein C-terminus (SGSPXARR, X is M or V) in human, bovine, rabbit, and chicken. None of these phosphorylated sites were detected in spiny dogfish and appear to be absent also in rattlesnake as no clear evidence of phosphorylation was found from the intact mass analysis and de novo sequencing study (see below).
Deamidated Q102 was found to be present in components C2, C3, C8a, and C8b (Figure S10
); A deamidated glutamine at position 120 is also presented in C3 (). Deamidation of Q102 and Q120 was also observed in human MBP but has not been examined for rabbit MBP8,9
. Overall, the C1 fraction was least modified while the less basic C3 and C8b fractions were more highly modified. The analysis of proteolyzed MBP fractions were also consistent with our intact mass analysis of MBP proteins that the deconvoluted spectra showed C3 had the most complicated spectra as compared with C1 and C2 fractions(). We have no unambiguous evidence that rattlesnake MBP is deamidated and note that at least some of the Gln and Asn residues reported to be deamidated in mammals are substituted in rattlesnake by other residues. However, the MALDI MSMS sequence for the peptide isolated from a GluC digest having the sequence HKYAHQ/KGHQ/KGYRYDGE appears to end in a Glu residue despite strong evidence for a Gln residue in other peptides spanning this region.
The oxidized methionine in bovine MBP at position 19 has not been reported before and was found in all five components in this study (Figure S11
). While it is quite likely that the methionine oxidation observed in this study resulted after the protein was extracted from the cell, we cannot rule out its presence in vivo
. It is worth noting that several studies in other proteins have demonstrated that methionine oxidation has a significant effect on protein function, stability, aggregation and folding34–38
. More generally, oxidation of methionine can decrease protein stability. One explanation for the effect is that the extra oxygen atom introduced by methionine oxidation changes the protein hydrophobicity at that site39
Intact mass study of bovine MBP charge components and unfractionated rattlesnake MBP
The presence and distribution of several PTMs in three charge components of bovine MBP, C1–3, have been successfully characterized by intact mass analysis(). In C1, the first major component had a calculated monoisotopic mass of 18,354.44 Da while the theoretical monoisotopic mass calculated from the protein sequence was 18312.39 Da. The mass difference was +42.05 Da which is consistent with the N-terminal acetylation observed at the peptide level. The mass of the second major peak was further increased by 14.04 Da which is consistent with monomethylation of the N-terminally acetylated protein. The third major peak had a mass difference of +28.05 Da from the first peak suggesting that MBP in the C1 fraction was also modified with a dimethylation. The most abundant peak (Mw=18,368.48 Da) in the C1 fraction corresponds to MBP modified with both N-terminal acetylation and monomethylation. All the ions found in the C1 fraction were also present in the C2 fraction. However, the most abundant peak in C2 corresponds to the protein modified with N-terminal acetylation and dimethylation. An additional species mass shifted by +0.99 Da from 18,368.48 Da was found in C2, which is consistent with either deamidation or citrullination. Evidence consistent with the presence of an oxidized Met in C2 came from a peak with mass increased by +16 Da from 18,382.48 Da. The C3 component had the most complex spectrum arising from the combination of different PTMs. In addition to those PTMs found in C1 and C2, deamidated or citrullinated residues were suggested by mass increases of 1 or 2 Da. Although all three components were modified with a mono- and di-methylated Arg, the distribution of non-methylated, mono- and dimethylated forms changed in different charge isomers. For instance, the ratio of non-methylated: monomethylated: dimethylated was 1 :1.18 :0.80 in C1 while this ratio was changed to 1 :0.45:1.14 in C2. Generally, the results from intact mass analysis were consistent with those from analysis of the proteolytic digests: C1 was least modified while C3 was most modified.
Unlike the spectra from bovine MBP fractions C1–C3, intact mass analysis of rattlesnake MBP revealed a much simpler spectrum (), suggesting rattlesnake MBP has considerably fewer PTMs than bovine MBP although the protein sequences are fairly well conserved in many species. Note that the complete protein sequence for rattlesnake MBP is unknown. The intact monoisotopic mass of rattlesnake MBP was approximately 19,564 Da. A form having a mass shift of 1 Da was observed for intact rattlesnake MBP suggesting rattlesnake MBP might be deamidated or citrullinated. This is consistent with the evidence with citrullination observed from the bottom-up analysis discussed below. No evidence was observed for deamidation in the bottom-up analysis. A mass shift of 14 Da is consistent with the bottom-up analysis which identified methylation of Arg in the peptide, GRGLSFR. Spectra of intact rattlesnake MBP are generally less post-translationally modified compared with spectra of intact bovine MBP.
Partial protein sequence of rattlesnake MBP and its PTMs
We performed Mascot database searching against the entire protein sequence database with error tolerance as well as de novo
sequence analysis of peptides from rattlesnake MBP to search for conserved sequences and possible PTMs present in the protein. The de novo
sequencing software, PEAKS 5.3, was employed in this study for Orbitrap with manual confirmation. The error tolerance was 10ppm for precursor ions and 0.8 Da for fragment ions. Total Local Confidence (TLC) and Average Local Confidence (ALC%) scores have been used to determine the quality of the predicted sequence for particular MS/MS spectra40
. In addition to these procedures, independent manual de novo
sequencing was performed on MALDI TOFTOF MSMS spectra by an expert (Walker). Mascot database searching together with the de novo
interpretations identified more than 20 unique peptides (or a total of 150 AAs) and 4 PTMs (acetylation, monomethylation, dimethylation, deimination) at 4 sites in rattlesnake MBP (Table S1
, Figure S12
), which covers 85% of gekko and 89% of anole MBP sequences respectively (). One additional potential modification was observed for deamidation of Gln to generate the C-terminal Glu residue in the peptide HKYAHKGHKGYRYDGE. The residue assignments for I/L and K/Q in were based on the Anole and Gekko sequences except when v and w fragment ions were able to distinguish I/L (See Figure S13
for an annotated example spectrum and Table S1
under MALDI TOFTOF peptides. The sequence of rattlesnake MBP exhibited good sequence identity with most species but is closest to reptile MBP sequences (gekko and anole) (). Mono- and dimethylated Arg were also identified in a peptide with the sequence GR
GLSFSR and this peptide exhibited a very similar fragmentation pattern to the homologous peptide from bovine MBP having only a single amino acid difference (). Other PTMs found in this species are: Protein N-terminal acetylation in a chymotryptic peptide (Figure S8
) and a deiminated Arg in two tryptic peptides (LATASTIDHAR
HGSPR and GYR
YDGQGTLSK) (Figures S5 and S6
). Both of these sites are partially deiminated as evidenced by observation of peptides containing arginine as well as peptides containing citrulline. All MBPs from other species including rattlesnake, which have been previously investigated or addressed in this study, have been found to have acetylated alanine at the N-terminus indicating this modification is conserved across different species ().
Rattlesnake MBP partial sequence and PTMs matched to other species