Deamidation of -Asn-Gly- Sequences during Sample Preparation for Proteomics:  Consequences for MALDI and HPLC-MALDI Analysis

Abstract

We find that peptides containing -Asn-Gly- sequences typically show approximately 70-80% degree of deamidation after standard overnight (approximately 12 h) tryptic digestion at 37 degrees C. This emphasizes the need for more detailed information about the deamidation reaction in -Asn-Gly- sequences, in which two deamidated species are produced, one containing an aspartic acid (-Asp-Gly-) residue and the other containing an isoaspartic acid (-betaAsp-Gly-) residue. For the peptide SLNGEWR (54-60 beta-galactosidase, E. coli), all three components of the reaction mixture were separated by HPLC on C18 300-A sorbent, with trifluoroacetic acid as an ion-pairing modifier. Their intensity ratios suggested the elution order -betaAsp-/-Asn-/-Asp-, which was subsequently confirmed by MALDI MS and MS/MS analysis. The kinetics of the deamidation was studied in detail for the synthetic SLNGEWR parent using RP HPLC with UV detection. The half-life of this peptide was found to be approximately 8 h under digestion conditions. Analysis of a large pool of peptide retention data shows that the -betaAsp-/-Asn-/ -Asp- retention order is normally observed under the above conditions, especially if the original -NG- sequence is surrounded by hydrophobic amino acids. However, changing chromatographic conditions to 100-A pore size sorbents, or using formic acid as a modifier, increases the retention time of -betaAsp- relative to the -Asn-/-Asp- pair, so the order can sometimes be different.

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

“…Deamidation of Asn, most common in -Asn-Gly sequences, yields Asp and bAsp and is usually observed in proteomic workflows involving overnight tryptic digestion (Krokhin et al, 2006). Two peaks were observed for the deamidated species (Asp and bAsp are isomeric) of P5-1 and one species eluted closely with the unmodified P5-1 (not shown).…”

“…Two peaks were observed for the deamidated species (Asp and bAsp are isomeric) of P5-1 and one species eluted closely with the unmodified P5-1 (not shown). Previous studies revealed that reversed-phase LC using FA as an ion-pairing modifier separated native and deamidated peptides in the order of -Asn2/2bAsp2/2Asp- (Krokhin et al, 2006). In addition, the only reference peptide for quantification of the ClpP3 subunit (P3-2) contains a Met, which can readily undergo various states of oxidation during sample preparation.…”

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

“…For example, under physiological conditions, linear peptides with -NI-were shown to have a deamidation half-life of 300 d opposed to peptides with -NG-that had a half-life of 1 d [10]. Therefore, deamidation of -NG-is a common peptide modification that results from in-gel digestion protocols that require overnight incubation in ammonium bicarbonate (ϳpH 8.5) with trypsin [12,13]. Asp isomerization is also possible (Figure 1), but occurs ϳ40 times slower than deamidation [2] and is therefore a less prevalent modification.…”

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

“…It is known that in-solution digestion by trypsin can induce artificial modifications such as asparagine deamidation (Krokhin et al, 2006) and N-terminal glutamine cyclization (Bongers et al, 1992;Dick et al, 2007) on target protein due to the elevated temperature and alkaline pH buffers used during digestion for overnight. Proteolysis by our protease-immobilized microreactors was achieved within a short period of time (~ 20 min) at 30 C therefore suggesting these artificial modifications as a remote possibility.…”