Detection, Evaluation and Minimization of Nonenzymatic Deamidation in Proteomic Sample Preparation

Abstract

Identification of deamidated sites in proteins is commonly used for assignment of N-glycosylation sites. It is also important for assessing the role of deamidation in vivo. However, nonenzymatic deamidation occurs easily in peptides under conditions commonly used in treatment with trypsin and PNGase F. The impact on proteomic sample preparation has not yet been evaluated systematically. In addition, the 13 C peaks of amidated peptides can be misassigned as monoisotopic peaks of the corresponding deamidated ones in database searches. The 19.34 mDa mass difference between them is proposed as a means for eliminating the resulting false positive identifications in large-scale proteomic analysis. We evaluated five groups of proteomic data, obtained mainly through an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)-reverse phase (RP) chromatography sequence, and ascertained that nonenzymatic asparagine deamidation occurred to some extent on 4 -9% of the peptides, resulting in the false positive identification of many N-glycosylation sites. A comprehensive investigation indicated that the chief causative factors were the mildly alkaline pH and prolonged incubations at 37°C during proteomic sample preparation. An improved protocol is proposed featuring tryptic digestion at pH 6 and deglycosylation at pH 5, resulting in a significant decrease in nonenzymatic deamidation while conserving adequate digestion efficiency. The number of identified deamidation sites was improved significantly by increasing the sample loading amount in liquid chromatography-tandem MS. This permitted the identification of a significant number of glutamine deamidation sites, which featured sequence motifs largely different from those for asparagine deami- Accurate identification of protein deamidation sites is important to understanding the role of deamidation in human diseases and aging and in the correct location of N-glycosylation using the PNGase F deglycosylation method. However, determination of protein deamidation with liquid chromatography-tandem MS (LC-MS/MS) 1 is compromised by current methods of proteomics sample preparation and database searching. Nonenzymatic deamidation of Asn and Gln occurs spontaneously on proteins and peptides both in vivo and in vitro. In vivo, deamidation is reported to be related to Alzheimer's disease and cataracts (1-3). It has also been proposed as a molecular clock in studies of aging (4, 5). In vitro, deamidation leads to the degradation of therapeutic proteins during production and storage (6, 7). It takes place relatively slowly in intact proteins with a half-life of 1-500 days for Asn and 100 -500 days for Gln (8). However, the deamidation rate of peptides increases significantly under conditions typical of tryptic digestion, i.e. incubation in digestion buffers at pH 8 for 8 -16 h at 37°C (9). Under mildly alkaline conditions, Asn deamidation happens mainly through the formation of a succinimide ring intermediate that is quickly hydrolyzed to D,LAsp and D,L-isoAsp with isoAsp pred...

Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431

“…To further confirm the quality of the identification, a manual check for the spectrum of each glycopeptide was performed. Sometimes database searches return some false-positive identifications of deamidated peptides because the 13 C peaks of amidated peptides can be wrongly assigned as the monoisotopic peaks of the corresponding deamidated peptides (56). The close N or Q to the motif of glycosylation can also cause false-positive identification if the evidence is not enough to confirm which one is really deamidated.…”

“…Nonenzymatic deamidation occurs in peptides under basic conditions commonly used in treatment with trypsin and PNGase F. Our previously published results showed that digesting peptides in pH 6.0 and releasing glycans in pH 5.0 significantly reduced the artifacts of glycosite identification from deamidated peptides introduced in sample preparation (56). Solutions with pH 6.0 and with pH 5.0 were used to extract A431 proteins and remove glycans of peptides after enrichment, respectively.…”

A Novel Method for the Simultaneous Enrichment, Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development

“…Nonenzymatic deamidation has previously been observed during the extended incubations with either trypsin or PNGase F digestions (35). We have previously demonstrated that false positive glycosylations may occur in large-scale datasets by mis-annotation of chemically deamidated Asn residues within an N-linked sequon (21,22).…”

“…This gives rise to an approximate 1.2% glycosite FDR (39/3246) (supplemental Table S6). A further improvement to these negative control experiments could include the combination of an optimized trypsin and PNGase F protocol described in (35) with or without incubation of the tryptic peptides at increased pH and temperature to observe the formation of deamdiated peptides.…”

Advances in purification and separation of posttranslationally modified proteins

“…The rates are significantly higher at alkaline pH, which is typical (for instance) in trypsin digestion. Thus, it is difficult to distinguish a natural protein deamidation, which could be a marker of a disease or protein ageing, from artifactual deamidation during proteomic sample preparation [201,202]. Deamidation proceeds via formation of a cyclic intermediate by condensation of the side-chain amide group with the carbonyl group of the following amino acid, and subsequent opening of this ring upon hydrolysis with water.…”