Peptide Mapping Research Articles

Coupling Anion Exchange Chromatography with Native Mass Spectrometry for Charge Heterogeneity Characterization of Monoclonal Antibodies.

Despite the recent success of coupling anion exchange chromatography with native mass spectrometry (AEX-MS) to study anionic proteins, the utility of AEX-MS methods in therapeutic monoclonal antibody (mAb) characterization has been limited. In this work, we developed and optimized a salt gradient-based AEX-MS method and explored its utility in charge variant analysis of therapeutic mAbs. We demonstrated that, although the developed AEX-MS method is less useful for IgG1 molecules that have higher isoelectric points (pIs), it is an attractive alternative for charge variant analysis of IgG4 molecules. By elevating the column temperature and lowering the mAb pI through PNGase F-mediated deglycosylation, the chromatographical resolution from AEX separation can be significantly improved. We also demonstrated that, after PNGase F and IdeS digestion, the AEX-MS method exhibited excellent resolving power for multiple attributes in the IgG4 Fc region, including unprocessed C-terminal Lys, N-glycosylation occupancy, and several conserved Fc deamidations, making it ideally suited for multiple attribute monitoring (MAM). Through fractionation and peptide mapping analysis, we also demonstrated that the developed AEX-MS method can provide site-specific and isoform-resolved separation of Fc deamidation products, allowing rapid and artifact-free quantitation of these modifications without performing bottom-up analysis.

The Importance of the C-Terminal Cys Pair of Phosphoribulokinase in Phototrophs in Thioredoxin-Dependent Regulation.

Phosphoribulokinase (PRK), one of the enzymes in the Calvin–Benson cycle, is a well-known target of thioredoxin (Trx), which regulates various enzyme activities by the reduction of disulfide bonds in a light-dependent manner. PRK has two Cys pairs conserved in the N-terminal and C-terminal regions, and the N-terminal one near the active site is thought to be responsible for the regulation. The flexible clamp loop located between the N-terminal two Cys residues has been deemed significant to Trx-mediated regulation. However, cyanobacterial PRK is also subject to Trx-dependent activation despite the lack of this clamp loop. We, therefore, compared Trx-mediated regulation of PRK from the cyanobacterium Anabaena sp. PCC 7120 (A.7120_PRK) and that from the land plant Arabidopsis thaliana (AtPRK). Interestingly, peptide mapping and site-directed mutagenesis analysis showed that Trx was more effective in changing the redox states of the C-terminal Cys pair in both A.7120_PRK and AtPRK. In addition, the effect of redox state change of the C-terminal Cys pair on PRK activity was different between A.7120_PRK and AtPRK. Trx-mediated redox regulation of the C-terminal Cys pair was also important for complex dissociation/formation with CP12 and glyceraldehyde 3-phosphate dehydrogenase. Furthermore, in vivo analysis of the redox states of PRK showed that only one disulfide bond is reduced in response to light. Based on the enzyme activity assay and the complex formation analysis, we concluded that Trx-mediated regulation of the C-terminal Cys pair of PRK is important for activity regulation in cyanobacteria and complex dissociation/formation in both organisms.

Development and certification of a pharmacopoeial reference standard for primary structure identification of purified recombinant interferon beta-1b by peptide mapping

Medicines based on recombinant human interferons (rhIFNs) beta-1a and beta-1b are used as first-line treatment of multiple sclerosis. Meanwhile, rhIFN beta-1a and beta-1b have structural differences associated with the eukaryotic or prokaryotic expression systems, respectively. Pharmacopoeias require identification of the primary structure of recombinant proteins by peptide mapping, which involves the use of reference material. Currently, there is no international reference standard available for rhIFN beta-1b structural identification. The aim of the study was development and certification of a pharmacopoeial reference standard for identification of the amino acid sequence of purified rhIFN beta-1b by peptide mapping. Materials and methods: rhIFN beta-1b produced by GENERIUM and endoproteinase Glu-C from Staphylococcus aureus V8 were used in the study. The peptide mapping was performed using reverse-phase high-performance liquid chromatography (RP HPLC) and high-resolution mass spectrometry. Statistical evaluation of the results included calculation of the arithmetic mean, standard deviation, and coefficient of variation. Results: the authors developed and certified a Russian Pharmacopoeia reference standard for structural identification of rhIFN beta-1b (PhRS 3.2.00447). The certified characteristic is the range of retention times of characteristic peaks: the absolute retention time was 42.0–43.2 for the third (reference) peak, the relative retention time was 0.61–0.66 for the first peak, 0.68–0.73 for the second peak, 1.04–1.06 for the fourth peak, 1.14–1.15 for the fifth peak, 1.22–1.24 for the sixth peak, and 1.29–1.30 for the seventh peak. Conclusions: the authors developed requirements for the rhIFN beta-1b pharmacopoeial reference standard. The material chosen as the candidate reference standard was an intermediate rhIFN beta-1b product sampled before addition of human serum albumin. The quality control was carried out in accordance with the developed specification. The authors analysed the amino acid sequence of the molecule, confirmed the presence of the disulfi e bond, and obtained the certifi d characteristic of the reference standard. Comparative analysis of the peptide maps of the certified rhIFN beta-1b pharmacopoeial reference standard and the rhIFN beta-1a reference standard revealed differences between the maps, and, therefore, confirmed the relevance of the developed reference standard.

At-line quantitative profiling of monoclonal antibody products during bioprocessing using HPLC-MS

(1) Background: The N-glycosylation profile as well as the subunit assembly of monoclonal antibodies (mAbs) are strongly dependent on manufacturing conditions and thus need to be monitored during the bioprocess. Commonly, mAbs are characterized downstream of the fermentation process applying different analytical techniques like released glycan analysis, peptide mapping, or subunit profiling. However, these procedures are time-consuming and difficult to perform in real-time. (2) Methods: We applied a simple HPLC-MS workflow with minimal sample preparation to characterize mAb product quality at the intact protein level at different time points during fermentation. After harvest, the cell culture medium was centrifuged briefly. The supernatant containing the fermentation product was diluted and immediately subjected to HPLC-MS analysis. (3) Results: Besides the product of interest (mAb), the fermentation broth contained misassembled variants, mostly light chain and light chain dimer. The mAb's glycosylation profile changed over time showing an increase in galactosylated variants with G0F/G1F being the most abundant glycoform at all time points of fermentation. Furthermore, expressed protein species were relatively and absolutely quantified. The workflow was very robust despite analyzing a highly complex matrix. Relative standard deviations for retention times were below 0.5% for both intra and inter-day comparison, whereas relative procedural standard deviations for quantification of the different protein species ranged between 7 and 13%. (4) Conclusions: This approach allows for reliable analysis of product profiles of monoclonal antibody species including misassembled subunits and glycosylation variants directly from fermentation broth using a fast and robust HPLC-MS workflow.

Dendrimer-Modified Silica Nanoparticles for Efficient Enrichment of Low-Concentration Peptides

Peptide profiling based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is of particular interest as it can provide physiologically and pathologically related information of the bio-samples. Due to the complexity of real biological samples, MALDI-TOF MS-based peptide mapping methods rely strongly on particular enrichment methods to improve the signal intensity. This paper introduces third-generation dendrimer-modified SBA-15 with the surface functionalization of amino and carboxyl group, respectively (denoted as SBA-15/G3-NH2 and SBA-15/G3-COOH), for the efficient capture of low-abundance peptides. The enrichment ability of the nanocomposites was evaluated by standard peptides digests and real biological samples. The synthesized nanocomposites incorporated the benefit of dendrimers and mesoporous silica nanomaterial SBA-15, showing enhanced peptide enrichment ability. Therefore, this work may provide a new class of nanomaterials for peptide mapping from biological samples.

Methodology for Analysis of Peptide Consumption by Yeast during Fermentation of Enzymatic Protein Hydrolysate Supplemented Synthetic Medium Using UPLC-IMS-HRMS

Several studies have shown the ability of yeast to consume peptides as a nitrogen source in single-peptide containing media. However, a suitable and cost-effective methodology to study the utilization of peptides by yeast and other microorganisms in a complex peptide mixture has yet to be put forward. This article addresses this issue by presenting a screening methodology for tracking the consumption of peptides by yeast during alcoholic fermentation. As a peptide source, the methodology makes use of an in-house prepared peptide-mapped bovine serum albumin (BSA) proteolytic digest, which was applied to a synthetic grape must. The peptide uptake was analyzed using high-throughput ultra-high-pressure liquid chromatography coupled to data-independent acquisition-based ion mobility separation-enabled high-resolution mass spectrometry (UPLC-DIA-IMS-HRMS) analysis. The relative changes of abundance of 123 di- to hexapeptides were monitored and reported during fermentations with three commercial wine strains, demonstrating different uptake kinetics for individual peptides. Using the same peptide-mapped BSA hydrolysate, the applicability of an untargeted workflow was additionally assessed for peptide profiling in unelucidated matrixes. The comparison of the results from peptide mapping and untargeted analysis experiments highlighted the ability of untargeted analysis to consistently identify small molecular weight peptides on the length and amino acid composition. The proposed method, in combination with other analytical techniques, such as gene or protein expression analysis, can be a useful tool for different metabolic studies related to the consumption of complex nitrogen sources by yeast or other microorganisms.

Effect of fruit maturation on N-glycosylation of plant-derived native and recombinant miraculin

Miracle fruit, Synsepalum dulcificum, produces a unique taste-modifying protein, miraculin (MIR), which has an attractive potential for commercial application as a novel low-calorie sweetener. To establish a stable supply system for MIR, a previous study established a platform for recombinant MIR (rMIR) production in tomato plants and demonstrated that native miraculin from miracle fruit (nMIR) and rMIR were almost identical in their protein modifications with N-glycan. However, neither N-glycosylation nor the influence of fruit maturation on the structural changes of N-glycan have been fully characterized in detail. Here, with a focus on N-glycosylation and the contribution of fruit maturation to N-glycan, we reanalyzed the N-glycosylation of the natural maturation stages of nMIR and rMIR, and then compared the N-glycan structures on MIRs prepared from the fruit at two different maturation stages. The detailed peptide mapping and N-glycosylation analysis of MIRs provided evidence that MIRs have variants, which were derived mainly from the differences in the N-glycan structure in nMIR and the N-glycosylation in rMIR and not from the cleavage of the peptide backbone. N-Glycan analysis of MIRs from the maturation stage of fruits demonstrated that N-glycan structures were similar among nMIRs and rMIRs at every maturation stage. These results indicated that the heterogeneously expressed rMIRs had the same characteristics in post-translational modifications, especially N-glycosylation and N-glycan structures, throughout the maturation stages. This study demonstrated the potential of recombinant protein expressed in tomato plants and paves the way for the commercial use of rMIR.

A Reference Standard for Analytical Testing of Erythropoietin

PurposeErythropoietin (EPO) is a 165 amino acid protein that promotes the proliferation of erythrocytic progenitors. A decrease in endogenous EPO production causes anemia that can be treated with recombinant Human EPO (rHuEPO).ObjectiveTo ensure the safety and efficacy of the rHuEPO, manufacturers must use analytical methods to demonstrate similarity across batches and between different products. To do this they need reference standards to validate their equipment and methods.MethodWe used peptide mapping, size-exclusion chromatography, glycoprofiling, and isoelectric focusing to analyze a rHuEPO reference standard.ResultsCharacterization demonstrates that our rHuEPO reference standard meets the criteria for quality.ConclusionThe rHuEPO reference standard is fit for purpose as a tool for validating system suitability and methods.

Glycation Alters the Fatty Acid Binding Capacity of Human Serum Albumin.

Glycation significantly alters the physicochemical and biofunctional properties of proteins in foods and in vivo. In the present study, human serum albumin (HSA) as the major transporter of fatty acids was modified with glyoxal under physiological conditions. Reversibly albumin-bound glyoxal was removed, and advanced glycation end products were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total modification of protein-bound lysine and arginine residues reached up to 4.2 and 9.6%, respectively. The impact of these modifications on the transport capacity of long-chain fatty acids was characterized by spin-labeled fatty acid probes via electron paramagnetic resonance spectroscopy. With increasing degree of glycation, the equivalence of the seven binding sites of native HSA with a dissociation constant of 0.74 ± 0.09 μM was set off with only the three high-affinity sites 2, 4, and 5 remaining (0.46 ± 0.07 μM). The other four sites were shifted to low affinities with significantly higher dissociation constants (1.32 ± 0.35 μM). Tryptic peptide mapping enabled us to relate these findings to molecular changes at specific binding sites. Modification hotspots identified were lysine 351, 286, 159 and arginine 144, 485, 117. Further investigation of plasma protein samples of uremic patients vs healthy controls gave first insights into the in vivo situation.

A Mass Spectrometric Characterization of Light-Induced Modifications in Therapeutic Proteins

During the development of a therapeutic protein, its quality attributes that pertain to the primary structure must be appropriately characterized, commonly by LC-MS/MS peptide mapping experiments. Extracting attribute information from LC-MS/MS data requires knowledge of the attribute of interest. Therefore, it is important to understand all potential modifications on the therapeutic proteins. In this work, we performed UV and visible light irradiation experiments on several therapeutic proteins, with or without the presence of a photosensitizer. Light-induced modifications were detected and characterized by tryptic digestion followed by LC-MS/MS analysis. A list of potential light-induced modifications, with their respective mass changes, was obtained. These modifications are primarily on methionine, tryptophan, histidine, cysteine, tyrosine and phenylalanine residues. Many of these modifications have not been previously reported on therapeutic proteins. Our findings therefore provide a database of potential light-induced modifications that would enable the routine characterization of light-induced modifications on therapeutic proteins.

Characterization of the Reduced IgE Binding Capacity in Boiled and Autoclaved Soybeans through Proteomic Approaches.

The study investigated the changes in IgE binding capacity, protein profiles and peptide compositions after soybeans were boiled and autoclaved. The results of ELISA showed that the IgE binding capacity of soybean was reduced by 69.3% and 88.9% after boiling and autoclaving, respectively. Above 43 and 10 kDa proteins disappeared in boiled and autoclaved soybeans from SDS-PAGE, respectively. A Venn diagram and heat map showed that there was no change in allergen types and a reduction in allergen contents in the boiled and autoclaved soybeans. The changes in peptide compositions were also observed in the boiled and autoclaved soybeans through Venn diagram, PCA and heat map. LC/MS-MS and peptide mapping analysis demonstrated that boiling and autoclaving masked many epitopes in Gly m 4 and Gly m 5, such as ALVTDADNVIPK, SVENVEGNGGPGTIKK and KITFLEDGETK of Gly m 4 and VEKEECEEGEIPRPRPRPQHPER of Gly m 5, resulting in a reduction of IgE binding capacity in the extracted proteins. By contrast, the exposure of many epitopes in Gly m 6 was observed in boiled and autoclaved soybeans, which might be mainly responsible for the existing IgE binding capacity in the treated soybean proteins. Interestingly, the IgE binding capacity of soybeans showed a positive correlation with the total contents and number of peptides in Gly m 4–Gly m 6.

Site-Specific Conjugation Quantitation of a Cysteine-Conjugated Antibody-Drug Conjugate Using Stable Isotope Labeling Peptide Mapping LC-MS/MS Analysis.

Drug-load (DL) characterization of antibody-drug conjugates (ADCs) is an important analytical task due to its designation as a critical quality attribute (CQA) affecting potency and stability. Intact and subunit liquid chromatography-mass spectrometry (LC-MS) analyses can determine global drug-to-antibody ratios (DARs) that correlate well with other orthogonal analytical methods; however, peptide mapping liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has struggled to provide complementary site-specific quantitation of drug conjugation sites. The peptide mapping method described herein utilizes stable isotope labeling to accurately quantitate the site-specific conjugation levels of a cysteine-conjugated ADC to provide "bottom-up" DAR characterization in parallel with protein sequence and post-translational modification (PTM) characterization in one multi-attribute analytical method (MAM).

Physicochemical stability of PF-05280014 (trastuzumab-qyyp; TrazimeraTM), a trastuzumab biosimilar, under extended in-use conditions.

The stability and functional activity of the trastuzumab biosimilar PF-05280014 (trastuzumab-qyyp; TrazimeraTM), was assessed under extended in-use conditions. PF-05280014 was diluted in 0.9% sodium chloride to final concentrations of 0.2 mg/mL and 4 mg/mL in 3 different types of infusion bags (polyolefin, ethylene vinyl acetate, and polyvinyl chloride). Infusion bags containing diluted PF-05280014 were stored at 25 ± 5° C for 24 h, before storage at 5 ± 3° C for 0, 1, 2, 4, or 6 weeks. Following extended storage, samples of PF-05280014 were removed from the infusion bags and stored at 25 ± 5° C for 24 h before biophysical and functional characterization. In addition to the visual characteristics of each sample at the various time points, the stability of PF-05280014 was assessed using a variety of biophysical techniques, including size-exclusion high-performance liquid chromatography, non-reducing sodium dodecyl sulfate capillary electrophoresis, cation-exchange chromatography, peptide mapping, far-UV circular dichroism spectroscopy, and differential scanning calorimetry. The functional activity of PF-05280014 was evaluated using a cell-based growth inhibition assay. For all PF-05280014 concentrations, time points and infusion bags tested, there were no significant differences in visual characteristics or in protein concentration. The were no significant changes in the relative abundance of molecular weight or charge variants throughout the 6-week study period. Similarly, there were no significant changes in primary structure or in secondary structure content during the study. The relative potency of PF-05280014 was also maintained throughout the 6-week period. The stability and functional activity of PF-05280014 was maintained following dilution in 0.9% sodium chloride and storage for up to 6 weeks at 2-8° C.

Detailed Analytical Characterization of a Bispecific IgG1 CrossMab Antibody of the Knob-into-Hole Format Applying Various Stress Conditions Revealed Pronounced Stability.

In recent years, a variety of new antibody formats have been developed. One of these formats allows the binding of one type of antibody to two different epitopes. This can for example be achieved by introduction of the “knob-into-hole” format and a combined CrossMab approach. Due to their complexity, these bispecific antibodies are expected to result in an enhanced variety of different degradation products. Reports on the stability of these molecules are still largely lacking. To address this, a panel of stress conditions, including elevated temperature, pH, oxidizing agents, and forced glycation via glucose incubation, to identify and functionally evaluate critical quality attributes in the complementary-determining and conserved regions of a bispecific antibody was applied in this study. The exertion of various stress conditions combined with an assessment by size exclusion chromatography, ion exchange chromatography, LC–MS/MS peptide mapping, and functional evaluation by cell-based assays was adequate to identify chemical modification sites and assess the stability and integrity, as well as the functionality of a bispecific antibody. Stress conditions induced size variants and post-translational modifications, such as isomerization, deamidation, and oxidation, albeit to a modest extent. Of note, all the observed stress conditions largely maintained functionality. In summary, this study revealed the pronounced stability of IgG1 “knob-into-hole” bispecific CrossMab antibodies compared to already marketed antibody products.

Development of novel enzyme immobilization methods employing formaldehyde or triethoxysilylbutyraldehyde to fabricate immobilized enzyme microreactors for peptide mapping.

The digestion of proteins with proteolytic enzymes has expedited the analysis of peptide mapping. Here, we compared the digestion efficiency of soluble chymotrypsin (CT) with two immobilized CT preparations using bovine serum albumin (BSA) as the substrate. An efficient method of immobilizing chymotrypsin using formaldehyde (FA) was optimized and the conditions were applied to assess a novel immobilization reagent, triethoxysilylbutaraldehyde (TESB). Efforts to determine the best enzyme-to-substrate (E : S) ratios during digestion of denatured BSA with single-use FA-CT enzyme particles were performed by adjusting the amount of substrate used. An E : S ratio of 10 : 1 was found to be best based on the LC-MS/MS analysis data showing sequence coverage of 67%. Fabrication of immobilized enzyme microreactors (IMERs) was carried out using both (3-aminopropyl)triethoxysilane (APTES) with the idealized conditions with FA, as well as the novel procedure utilizing TESB for a proof of concept open-tubular IMER. It was found that the FA-APTES IMER had a sequence coverage of 6%, while the TESB IMER had 29% sequence coverage from MS analysis. The application of TESB in enzyme immobilization has the potential to facilitate a greater degree of enzymatic digestion with higher sequence coverage than traditional immobilization or crosslinking reagents for bottom-up proteomics.

Linchpin-directed precise labeling of lysine in native proteins, purification, and analysis.

The proteins are critical building blocks of living systems and serve as a tool for their investigation and intervention. Their precision engineering enables its tuning and expands the functional landscape. Among various proteinogenic amino acids, high-frequency lysine offers a promising bioconjugation target. However, it is also among the most challenging candidates for homogeneous single-site modification. The linchpin-directed modification (LDM) addresses this concern by offering chemoselective, site-selective, and modular protein bioconjugation. The protocol outlines a general method for single-site modification of a native protein. At first, the selected LDM reagent constructs a stable bioconjugate through acylation of the Lys side chain. Subsequently, its chemically orthogonal handle creates an opportunity to install desired probes directly. Alternatively, the same group enables bioconjugate enrichment through ordered immobilization. The subsequent release, coupled with probe installation, renders analytically pure single-site tagged protein bioconjugate. The analysis of these constructs involves intact MS of protein bioconjugate, peptide mapping, and MS-MS for the site of modification and homogeneity.

Rapid tryptic peptide mapping of human serum albumin using DI-MS/MSALL.

In recent decades, proteinic drugs, in particular monoclonal antibodies, are taking the leading role of small molecule drugs, and peptide mapping relying on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach to substitute the role of a ligand-binding assay for the quality control of the proteinic drugs. However, such LC-MS/MS approaches extensively suffer from time-intensive measurements, leading to a limited throughput. To achieve accelerated measurements, here, the potential of DI-MS/MSALL towards tryptic peptide mapping was evaluated through comparing with well-defined LC-MS/MS means, and human serum albumin (HSA) was employed as the representative protein for applicability illustration. Among the 55 tryptic peptides theoretically suggested by Skyline software, 47 were successfully captured by DI-MS/MSALL through acquiring the desired MS2 spectra, in comparison to 51 detected by LC-MS/MS. DI-MS/MSALL measurements merely took 5 min, which was dramatically superior to the LC-MS/MS assay. Noteworthily, different from fruitful multi-charged MS1 signals for LC-MS/MS, most quasi-molecular ions received lower charged states. DI-MS/MSALL also possessed advantages such as lower solvent consumption and facile instrumentation; however, more sample was consumed. In conclusion, DI-MS/MSALL is eligible to act as an alternative analytical tool for LC-MS/MS towards the peptide mapping of proteinic drugs, particularly when a heavy measurement workload.

Covalent Inhibition of Pyruvate Kinase M2 Reprograms Metabolic and Inflammatory Pathways in Hepatic Macrophages against Non-alcoholic Fatty Liver Disease.

Warburg effect of aerobic glycolysis in hepatic M1 macrophages is a major cause for metabolic dysfunction and inflammatory stress in non-alcoholic fatty liver disease (NAFLD). Plant-derived triterpene celastrol markedly inhibited macrophage M1 polarization and adipocyte hypertrophy in obesity. The present study was designed to identify the celastrol-bound proteins which reprogrammed metabolic and inflammatory pathways in M1 macrophages. Pyruvate kinase M2 (PKM2) was determined to be a major celastrol-bound protein. Peptide mapping revealed that celastrol bound to the residue Cys31 while covalent conjugation altered the spatial conformation and inhibited the enzyme activity of PKM2. Mechanistic studies showed that celastrol reduced the expression of glycolytic enzymes (e.g., GLUT1, HK2, LDHA, PKM2) and related signaling proteins (e.g., Akt, HIF-1α, mTOR), shifted aerobic glycolysis to mitochondrial oxidative phosphorylation and skewed macrophage polarization from inflammatory M1 type to anti-inflammatory M2 type. Animal experiments indicated that celastrol promoted weight loss, reduced serum cholesterol level, lipid accumulation and hepatic fibrosis in the mouse model of NAFLD. Collectively, the present study demonstrated that celastrol might alleviate lipid accumulation, inflammation and fibrosis in the liver via covalent modification of PKM2.

Development of a simple non-reduced peptide mapping method that prevents disulfide scrambling of mAbs without affecting tryptic enzyme activity

Non-reduced peptide mapping by liquid chromatography-mass spectrometry (LC-MS) analysis is a commonly used method for disulfide linkage characterization to assess structural integrity and quality of therapeutic monoclonal antibodies (mAbs). However, disulfide scrambling artifacts induced during sample preparation are often observed when basic pH and high temperatures are used during denaturation and digestion. To minimize disulfide scrambling artifacts, methods using various acidic pH conditions have been developed by multiple groups. However, lower pH conditions increase missed and non-specific cleavages, which complicates disulfide bond analysis because the majority of enzymes used in protein characterization are most efficient at alkaline pH. Here, we developed a non-reduced peptide mapping method for mAb characterization that minimizes disulfide scrambling at basic pH by adding an oxidizing agent, cystamine, and a low concentration of iodoacetamide (IAA) alkylating agent. Two human IgG1 mAbs, one with kappa light chain and another one with lambda light chain, were used as model proteins to develop and optimize the method. Using this novel method, disulfide scrambled peptides related to light chain-heavy chain (LC-HC) inter-disulfide disruption were significantly reduced with high reproducibility compared to conventional methods. Results demonstrated that the cystamine-added method is robust and minimizes disulfide scrambling artifacts produced during sample preparation.

Molecular Mapping of Urinary Complement Peptides in Kidney Diseases.

Defective complement activation has been associated with various types of kidney disease. This led to the hypothesis that specific urine complement fragments may be associated with kidney disease etiologies, and disease progression may be reflected by changes in these complement fragments. We investigated the occurrence of complement fragments in urine, their association with kidney function and disease etiology in 16,027 subjects, using mass spectrometry based peptidomics data from the Human Urinary Proteome/Peptidome Database. Twenty-three different urinary peptides originating from complement proteins C3, C4 and factor B (CFB) could be identified. Most C3-derived peptides showed inverse association with estimated glomerular filtration rate (eGFR), while the majority of peptides derived from CFB demonstrated positive association with eGFR. Several peptides derived from the complement proteins C3, C4 and CFB were found significantly associated with specific kidney disease etiologies. These peptides may depict disease-specific complement activation and could serve as non-invasive biomarkers to support development of complement interventions through assessing complement activity for patients’ stratification and monitoring of drug impact. Further investigation of these complement peptides may provide additional insight into disease pathophysiology and could possibly guide therapeutic decisions, especially when targeting complement factors.