Greg Grant described the compilation that he and Mark Crankshaw (Washington University, St. Louis) assembled entitled "Identification of Modified PTH-Amino Acids in Protein Sequence Analysis". This booklet includes most known natural as well as chemically modified residues using the ABI chromatography system and shows the elution positions of some residues using the Beckman-Porton instrument. Sequencing artifacts and the elution positions of some PTC-amino acids are also given, as are side-chain protected residues from the Boc and Fmoc peptide synthesis chemistries. The audience was encouraged to send chromatograms showing additional modified amino acids to be included in an updated version.
Some applications of sequencing to assign modifications were described by Reed Harris (Genentech). Blank cycles resulting from Nglycosylation are followed two residues later in the sequence by Thr, Ser or (rarely) Cys. Positive assignment of this glycosylation can be made after release of the N-glycan by peptide:N-glycosidase F (PNGase F, often referred to as N-glycanase), converting the attachment site to Asp (1). Release of oligomannose or hybrid-type N-glycans with endoglycosidase F (Endo F) will leave one GlcNAc residue attached to the glycosylated Asn. Deglycosylation by trifluoromethanesulfonic acid treatment (2) will also leave one GlcNAc residue attached to the glycosylated Asn. Asn(GlcNAc) can be obtained from Sigma (catalog number A 6681). PTH Asn(GlcNAc) can be identified by its elution between DTT and PTH-Asp. Serial digestion of a glycopeptide with Endo H, then PNGase F, can be used to distinguish Asn residues that are glycosylated with complex versus oligomannose/hybrid N-glycans [e.g., rgp120 (3)].
Most types of O-glycosylation will result in blank sequencing cycles. Consensus sequence(s) for the common GalNAc-linked ("mucin-type") O-glycans are under investigation and were discussed by Andrew Gooley; they are usually found near proline residues. Mucin-type structures can be released by a combination of neuraminidase and O-glycanase digestion (commercial kits are available from Oxford GlycoSystems and Glyko), so re-sequencing after such treatment will reveal the previously glycosylated Thr or Ser residue. Xylosylglucose glycans attached to Ser residues are found within Cys-Xaa-blank-Xaa-Pro-Cys sequences within EGF domains (4); no enzymatic approach to release such glycans has been reported. Gly-cosaminoglycans (common in the proteoglycans) are attached to serines that are followed by Gly residues. Fucosyl modifications are acid-labile, thus may not be detected as a blank sequencing cycle; O-linked fucose can be directly assigned only if the modified residue is within the first 5-6 cycles [e.g., tPA, factor XII (5)]. Sites of O GlcNAc modification have been identified by radiolabeling or MS/MS approaches, but it may be possible to assign such sites by Abernethy's approach with Thr-GalNAc (6).
Hydroxylation of Lys and Pro residues is common in collagens; such sites are found in Hyl-Gly or Hyp-Gly sequences. Hyl at the Lys277 position of rtPA was observed by recovery of the PTH-(e-PTC)-Hyl derivative, which elutes between PTH Val and DPTU on an ABI 120A system. b HydroxyAsp/Asn are found in Cys-Xaa-Hya/Hyn-(Xaa)4-Tyr/Phe-Xaa-Cys-Xaa-Cys sequences within EGF domains (7); these sites may be interpreted as blank cycles, because their PTH-derivatives elute in the RP-HPLC void.
Blocked N-termini are common in eukaryotic proteins. Digestion with pyroglutamate aminopeptidase will remove N-terminal pyroglutamate (Glp) residues (though it may also remove a Glp-Pro dipeptide), allowing sequencing to proceed. Similarly, an acylaminohydrolase can be used to remove N acetylated residues from peptides (8), though this enzyme prefers peptides with N acetylated Ala, Met, Thr, Ser, or Gly residues.
Cys residues can generate blank cycles several ways. Identification of the di PTH-cystine derivative was described by Mitsuru Haniu. Cys residues can also be involved in -CH2-S-CH2- lanthionine bonds (a synthetic peptide problem) (9), bonded to glutathione, or involved in intramolecular thioester bonds (e.g., complement C3 and C4, and a-2-macroglobulin).
Asn residues can deamidate spontaneously to form Asp and isoAsp, and Asp may isomerize to isoAsp; these processes generally occur via a succinimide intermediate in flexible regions of proteins and peptides. Isoaspartate will cause a complete loss of sequencing signal, while a succinimide will cause a partial signal reduction. The location of a stable succinimide can be determined by sequencing after alkaline hydroxylamine cleavage (10).
A number of protein modifications may not be detected during N-terminal sequence analysis, including Ser, Thr, Tyr, or Cys phosphorylation, Tyr sulfation, Cys acylation and g-carboxyglutamic acid. One way to ensure that protein modifications are not overlooked is to perform N-terminal sequence analysis on every fraction obtained from a RP-HPLC peptide map, then confirm the observed sequence(s) by mass spectrometry. A mass difference will reveal some information about the type of modification (11). The review by Aitken (12) details the consensus sequences known for most post-translational modifications.
Andrew Gooley presented work performed at Macquarie University (Sydney, Australia) using a Beckman-Porton solid-phase sequencer to study N- and O-linked glycosylation sites. The Beckman-Porton system permits the extraction and RP-HPLC analysis of glycosylated PTH-amino acids, allowing them to directly assign glycosylation sites [e.g., PsA and CD8a (13)]. The elution positions of Thr(HexNAc), Thr(GalNAc-Gal), Ser(GalNAc-Gal) and Asn(GlcNAc4-Man3-Gal0-2) were assigned. Two peaks are usually obtained for PTH-Thr(Sac). Glycoprotein/peptide samples must be desialated to prevent the sialic acid from forming a covalent bond with the support. PTH-glycoamino acids can be recovered for mass spectrometric or carbohydrate compositional analysis by high-pH anion-exchange chromatography, although high levels of background glucose may be present. Their studies indicated that mucin-type O glycosylation occurs within an Xaa-Pro-Xaa-Xaa motif, where Xaa is a glycosylated Thr or Ser (14).
Mitsuru Haniu (Amgen) discussed some examples of the recovery of di-PTH-cystine to aid in the assignment of disulfide bonds, as it is released only when the cycles containing both Cys residues have been sequenced. Di-PTH-Cys elutes just ahead of PTH-Tyr in ABI PTH-analysis systems. The recovery of di-PTH-Cys drops off quickly as a function of cycle number, with recoveries (relative to the peptide recovery) in the 20-70% range for cycles 1-7, then dropping below 20% after that. Recovery of di-PTH-Cys was sufficiently above background to detect disulfide bonds out to 20 cycles (15).
References
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