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Use of 18O labels to monitor deamidation during protein and peptide sample processing.

ABSTRACT: Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c. + 58 and z - 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)((n-1)+.) - 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of (18)O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (approximately 4 h) trypsin digestion process generates no additional detectable peptide deamidations.

SUBMITTER: Li X

PROVIDER: S-EPMC3105245 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Use of 18O labels to monitor deamidation during protein and peptide sample processing.

Li Xiaojuan X Cournoyer Jason J JJ Lin Cheng C O'Connor Peter B PB

Journal of the American Society for Mass Spectrometry 20080305 6

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each i ...[more]

PMID: 18394920

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Project description:High dose methotrexate (Mtx) therapy is commonly used in hemato-oncological practice. Alkalization of urine is a part of high dose methotrexate therapy for preventing crystallization in urine to avert renal insufficiency. Alkalization is monitored by urine pH at regular intervals. Oral pH has occasionally been used as a surrogate for oral mucositis. To compare and correlate pH of various body secretions (venous blood, oral salivary, lacrimal and urine) among patients undergoing alkalization with intravenous sodium bicarbonate during high dose methotrexate therapy. A prospective single center study in patients with hematological malignancies receiving Mtx > 1.5 g/m2 over 4-24 h. Patients were assessed for pH (from all 4 body fluids) at regular time intervals (q8 h) starting 6 h-prior and 48 h-post initiation of Mtx therapy. Mean pH of urine/oral was compared to surrogate samples. The mean oral pH was 6.9 (SD 0.65), the mean urinary pH was 7.59 (SD 0.773) the mean pH by venous blood gas analysis (venous pH) was 7.388 (SD 0.059), the mean lacrimal pH was 7.4536 (SD 0.527). Repeated measures ANOVA suggests that pH of different body fluids differ and cannot be used interchangeably [F (2.417, 309.361) = 54.89, p < 0.0005]. There was no statistically significant correlation between any other pair of assessed body fluids. On paired t test only the means of venous pH and urinary pH did not differ statistically (p 0.056). Venous pH significantly correlated with urinary pH but the strength of correlation was weak (r 0.184; p 0.037). pH of different body fluids is statistically different even when sampled simultaneously thus the pH of one fluid cannot be substituted for other. Based on this study we cannot substitute urinary pH with any other body fluids presently in patients undergoing high dose methotrexate and alkalization except in rare circumstances when venous pH can be used as a poor surrogate for urinary pH in situations where urinary pH cannot be monitored due to any reason. There was no surrogate for oral pH among the studied body fluids.

Dataset Information

Use of 18O labels to monitor deamidation during protein and peptide sample processing.

Publications

Use of 18O labels to monitor deamidation during protein and peptide sample processing.

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