Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.
ABSTRACT: Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH's (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.
Project description:A ubiquitous yet underappreciated protein post-translational modification, isoaspartic acid (isoAsp, isoD, or beta-Asp), generated via the deamidation of asparagine or isomerization of aspartic acid in proteins, plays a diverse and crucial role in aging, as well as autoimmune, cancer, neurodegeneration, and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 22.214.171.124) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of isoaspartate can be confirmed by electron transfer dissociation (ETD) mass spectrometry. As little as 0.5% of isoAsp was detected in synthetic beta-amyloid and cytochrome c peptides, even though both were initially assumed to be free of isoAsp. Taken together, our approach should expedite the unbiased discovery of isoAsp.
Project description:Due to its much slower deamidation rate compared to that of asparagine (Asn), studies on glutamine (Gln) deamidation have been scarce, especially on the differentiation of its isomeric deamidation products: alpha- and gamma-glutamic acid (Glu). It has been shown previously that electron capture dissociation (ECD) can be used to generate diagnostic ions for the deamidation products of Asn: aspartic acid (Asp) and isoaspartic acid (isoAsp). The current study explores the possibility of an extension of this ECD based method to the differentiation of the alpha- and gamma-Glu residues, using three human Crystallin peptides (alphaA (1-11), betaB2 (4-14), and gammaS (52-71)) and their potentially deamidated forms as model peptides. It was found that the z(*)-72 ions can be used to both identify the existence and locate the position of the gamma-Glu residues. When the peptide contains a charge carrier near its N-terminus, the c+57 and c+59 ions may also be generated at the gamma-Glu residue. It was unclear whether formation of these N-terminal diagnostic ions is specific to the Pro-gamma-Glu sequence. Unlike the Asp containing peptides, the Glu containing peptides generally do not produce diagnostic side chain loss ions, due to the instability of the resulting radical. The presence of Glu residue(s) may be inferred from the observation of a series of z(n)(*)-59 ions, although it was neither site specific nor without interference from the gamma-Glu residues. Finally, several interference peaks exist in the ECD spectra, which highlights the importance of the use of high performance mass spectrometers for confident identification of gamma-Glu residues.
Project description:Although differentiation of the isomeric Asn deamidation products (Asp and isoAsp) at the peptide level by electron capture dissociation (ECD) has been well-established, isoAsp identification at the intact protein level remains a challenging task. Here, a comprehensive top-down deamidation study is presented using the protein beta2-microglobulin (?(2)M) as the model system. Of the three deamidation sites identified in the aged ?(2)M, isoAsp formation was detected at only one site by the top-down ECD analysis. The absence of diagnostic ions likely resulted from an increased number of competing fragmentation channels and a decreased likelihood of product ion separation in ECD of proteins. To overcome this difficulty, an MS(3) approach was applied where a protein ion was first fragmented by collisionally activated dissociation (CAD) and the resulting product ion was isolated and further analyzed by ECD. IsoAsp formation at all three deamidation sites was successfully identified by this CAD-ECD approach. Furthermore, the abundance of the isoAsp diagnostic ion was found to increase linearly with the extent of deamidation. These results demonstrated the potential of ECD in the detection and quantitative analysis of isoAsp formation using the top-down approach.
Project description:Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn?Asp/IsoAsp conversions or Glu?Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.
Project description:Long-lived proteins are widespread in man, yet little is known about the processes that affect their function over time, or their role in age-related diseases.Racemization of two proteins from normal and cataract human lenses were compared with age using tryptic digestion and LC/mass spectrometry. Asp 151 in ?A crystallin and Asn 76 in ?S crystallin were studied.Age-dependent profiles for the two proteins from normal lenses were different. In neither protein did the modifications increase linearly with age. For ?A crystallin, racemization occurred most rapidly during the first 15 years of life, with approximately half of L-Asp 151 converted to D-isoAsp, L-isoAsp, and D-Asp in a ratio of 3:1:0.5. Values then changed little. By contrast, racemization of Asn 76 in ?S crystallin was slow until age 15, with isoAsp accounting for only 5%. Values remained relatively constant until age 40 when a linear increase (1%/year) took place. When cataract lenses were compared with age-matched normal lenses, there were marked differences in the time courses of the two crystallins. For ?A crystallin, there was no significant difference in Asp 151 racemization between cataract and normal lenses. By contrast, in ?S crystallin the degree of conversion of Asn 76 to isoAsp in cataract lenses was approximately double that of normals at every age.Modification of Asn and Asp over time may contribute to denaturation of proteins in the human lens. An accelerated rate of deamidation/racemization at selected sites in proteins, such as ?S crystallin, may contribute to cataract formation.
Project description: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.
Project description:The breakdown of long-lived proteins (LLPs) is associated with aging, as well as disease; however, our understanding of the molecular processes involved is still limited. Of particular relevance, cross-linked proteins are often reported in aged tissues but the mechanisms for their formation are poorly understood. In the present study, sites of protein cross-linking in human ocular lenses were characterized using proteomic techniques. In long-lived lens proteins, several sites of cross-linking were found to involve the addition of Lys to Asp or Asn residues. Using model peptides containing Asp or Asn, a mechanism was elucidated that involves a succinimide intermediate. Succinimides formed readily from Asn at neutral pH, whereas a higher rate of formation from Asp peptides was observed at more acidic pHs. Succinimides were found to be relatively stable in the absence of nucleophiles. Since racemization of Asp residues, as well as deamidation of Asn, involves a succinimide intermediate, sites of d-Asp and isoAsp in LLPs should also be considered as potential sites of protein covalent cross-linking.
Project description:Deamidation and the subsequent formation of isoaspartic acid (isoAsp) are common modifications of asparagine (Asn) residues in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar chemical properties and identical molecular mass. Recent studies showed that they can be differentiated using electron capture dissociation (ECD) which generates diagnostic fragments c'+57 and z•-57 specific to the isoAsp residue. However, the ECD approach is only applicable towards multiply charged precursor ions and generally does not work for ?-amino acids other than isoAsp. In this study, the potential of in-source decay (ISD) in characterization of isoAsp and other ?-amino acids was explored. For isoAsp-containing peptides, ISD with a conventional hydrogen-donating matrix produced ECD-like, c'+57 and z•-57 diagnostic ions, even for singly charged precursor ions. For other ?-amino acids, a hydrogen-accepting matrix was used to induce formation of site-specific a-14 ions from a synthetic ?-analogue of substance P. These results indicated that ISD can be broadly applied for ?-peptide characterization.
Project description:The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and nonenzymatic process, ubiquitous both in vivo and in nonbiological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC126.96.36.199) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. A total of 38 putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.
Project description:P. aeruginosa possesses the ability to utilize a wild range of compounds as the sole source of carbon and nitrogen, including proteogenic amino acids. In particular, utilization of L-Asp and L-Asn is insensitive to carbon catabolite repression as strong growth retains in the cbrAB mutants devoid of the essential regulators for the activation of most catabolic genes. Transcriptome analysis and functional characterization were conducted to identify genes that participate in the catabolism, uptake, and regulation of these two amino acids. Through gene knockout and growth phenotype analysis, degradation of L-Asn to L-Asp was shown to be mediated by two asparaginases AsnA and AsnB, whereas only AnsB is required for the deamidation of D-Asn to D-Asp. While D-Asp is a dead-end product, conversion of L-Asp to fumurate is catalyzed by an aspartase AspA as further evidenced by enzyme kinetics. The results from the measurements of promoter-lacZ expression in vivo and mobility shift assays in vitro demonstrated that the asnR and aspR genes encode two transcriptional regulators in response to L-Asn and L-Asp, respectively, for the induction of the ansPA operon and the aspA gene. In addition, exogenous L-Glu also cause induction of the aspA gene, most likely due to its conversion to L-Asp by the aspartate transaminase AspC. Expression of several transporters were also found inducible by L-Asn and/or L-Asp, including AatJQMP for acid amino acids, DctA and DctPQM for C4-dicarboxylates, and PA5530 for C5-dicarboxylates. In summary, a complete pathway and regulation for L-Asn and L-Asp catabolism was established in this study. Cross induction of three transport systems for dicarboxylic acids may provide a physiological explanation for the insensitivity of L-Asn and L-Asp utilization to carbon catabolite repression. Overall design: We conducted four independent microarray experiments in the presence (experimental) of L-Ala, L-Asn or L-Asp. P. aeruginosa PAO1 was grown aerobically in minimal medium P with 300 rpm shaking at 37°C, in the presence of 20mM Pyruvate with the addition of L-Ala, L-Asn or L-Asp at 20 mM.