Arginine Modifications by Methylglyoxal: Discovery in a Recombinant Monoclonal Antibody and Contribution to Acidic Species
Anal. Chem., Just Accepted Manuscript
Publication Date (Web): October 29, 2013
Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors, but still cannot completely account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal (MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to two adducts (dihydroxyimidazolidine and hydroimidazolone) with increase of molecular weights of 72 and 54 Daltons, respectively. In addition, the modification by methylglyoxal causes the antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to which an antibody was modified at multiple sites corresponds to the degree of shift in elution time. Furthermore, cell culture parameters also affected the extent of modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other metabolic pathways. Our findings again highlight the impact of cell culture conditions on the critical quality attributes, as we improve manufacturing processes via the quality by design (QbD) approach.
Discovery of Undefined Protein Crosslinking Chemistry: A Comprehensive Methodology Utilizing 18O-labeling and Mass Spectrometry
Anal. Chem., 2013, 85 (12), pp 5900–5908
Publication Date (Web): May 1, 2013
Characterization of protein crosslinking, particularly without prior knowledge of the chemical nature and site of crosslinking, poses a significant challenge due to their intrinsic structural complexity and the lack of a comprehensive analytical approach. Towards this end, we have developed a generally applicable workflow—XChem-Finder that involves four stages. (1) Detection of crosslinked peptides via 18O-labeling at C-termini. (2) Determination of the putative partial sequences of each crosslinked peptide pair using a fragment ion mass database search against known protein sequences coupled with a de novo sequence tag search. (3) Extension to full sequences based on protease specificity, the unique combination of mass, and other constraints. (4) Deduction of crosslinking chemistry and site. The mass difference between the sum of two putative full-length peptides and the crosslinked peptide provides the formulas (elemental composition analysis) for the functional groups involved in each cross-linking. Combined with sequence restraint from MS/MS data, plausible crosslinking chemistry and site were inferred, and ultimately, confirmed by matching with all data. Applying our approach to a stressed IgG2 antibody, ten cross-linked peptides were discovered and found to be connected via thioether originating from disulfides at locations that had not been previously recognized. Furthermore, once the crosslink chemistry was revealed, a targeted crosslink search yielded four additional crosslinked peptides that all contain the C-terminus of the light chain.
Chapter 287 - Peptidyl-Asp Metalloendopeptidase
Handbook of Proteolytic Enzymes (Third Edition), 2013, Pages 1281�1285Summary
The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 850 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 287 is Peptidyl-Asp Metalloendopeptidase.
An Integrated Proteomic Analysis of Major Isoaspartyl-Containing Proteins in the Urine of Wild Type and Protein L-Isoaspartate O-Methyltransferase-Deficient Mice
Anal. Chem., Just Accepted Manuscript
Publication Date (Web): January 17, 2013. Page: 85, 2423–2430
Copyright � 2013 American Chemical Society
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 non-enzymatic process, ubiquitous both in vivo and in non-biological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC184.108.40.206) 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. Thirty-eight 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.
SUBSTRATE-INDUCED CONTROL OF PRODUCT FORMATION BY PROTEIN ARGININE METHYLTRANSFERASE 1 (PRMT1)
Biochemistry. 2013 Jan 8;52(1):199-209. doi: 10.1021/bi301283t. Epub 2012 Dec 21.Abstract
Protein arginine methyltransferases (PRMTs) aid in the regulation of many biological processes. Accurate control of PRMT activity includes recognition of specific arginyl groups within targeted proteins, and also the generation of the correct level of methylation, none of which are fully understood. The predominant PRMT in vivo, PRMT1, has wide substrate specificity and is capable of both mono- and dimethylation, which can induce distinct biological outputs. What regulates the specific methylation pattern of PRMT1 in vivo is unclear. We report that PRMT1 methylates a multisite peptide substrate in a non-stochastic manner, with less C-terminal preference, consistent with the methylation patterns observed in vivo. With a single targeted arginine, PRMT1 catalyzed the dimethylation in a semi-processive manner. The degree of processivity is regulated by substrate sequences. Our results identify a novel substrate-induced mechanism for modulating PRMT1 product specificity. Considering the numerous physiological PRMT1 substrates, as well as the distinct biological outputs of mono- and dimethylation products, such fine-tuned regulation would significantly contribute to the accurate product specificity of PRMT1 in vivo and the proper transmission of biochemical information.
Chemical fingerprinting by RP-RRLC-DAD and principal component analysis of Ziziphora clinopodioides from different locations.
Nat Prod Commun. 2012 Sep;7(9):1181-4.Abstract
An efficient and accurate fingerprinting method using reversed-phase rapid-resolution liquid-chromatography coupled with photodiode array detection has been developed and optimized to examine the variance in active compounds among Ziziphora clinopodioides Lam from different locations. Three active components, diosmin, linarin and pulegone, were identified by matching their retention times and UV spectra with the corresponding reference compounds. Our results indicated that chromatographic fingerprints, in combination with principal component analysis (PCA) and hierarchical clustering analysis (HCA), could efficiently identify and distinguish Z. clinopodioides from different sources. Our fingerprinting methods and data will be useful for quality control, and thus, more effective dosing in clinical application of Z. clinopodioides.
Protein isoaspartate methyltransferase-mediated 18O-labeling of isoaspartic acid for mass spectrometry analysis.
Anal Chem. 2012 Jan 17;84(2):1056-62. Epub 2011 Dec 27.Abstract
Arising from spontaneous aspartic acid (Asp) isomerization or asparagine (Asn) deamidation, isoaspartic acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with (18)O via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 220.127.116.11) to the corresponding methyl ester, which is subsequently hydrolyzed in (18)O-water to regenerate isoAsp. The specific replacement of (16)O with (18)O at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a monoclonal antibody and the β-delta sleep-inducing peptide (DSIP) are demonstrated.