Proteomic aging clock (PAC) predicts age-related outcomes in middle-aged and older adults.

Beyond mere prognostication, optimal biomarkers of aging provide insights into qualitative and quantitative features of biological aging and might, therefore, offer useful information for the testing and, ultimately, clinical use of gerotherapeutics. We aimed to develop a proteomic aging clock (PAC) for all-cause mortality risk as a proxy of biological age. Data were from the UK Biobank Pharma Proteomics Project, including 53,021 participants aged between 39 and 70 years and 2923 plasma proteins assessed using the Olink Explore 3072 assay®. 10.9% of the participants died during a mean follow-up of 13.3 years, with the mean age at death of 70.1 years. The Spearman correlation between PAC proteomic age and chronological age was 0.77. PAC showed robust age-adjusted associations and predictions for all-cause mortality and the onset of various diseases in general and disease-free participants. The proteins associated with PAC proteomic age deviation were enriched in several processes related to the hallmarks of biological aging. Our results expand previous findings by showing that biological age acceleration, based on PAC, strongly predicts all-cause mortality and several incident disease outcomes. Particularly, it facilitates the evaluation of risk for multiple conditions in a disease-free population, thereby, contributing to the prevention of initial diseases, which vary among individuals and may subsequently lead to additional comorbidities.

MiR-98-5p plays suppressive effects on IL-1ß-induced chondrocyte injury associated with osteoarthritis by targeting CASP3.

BACKGROUND: This study aims to explore how miR-98-5p affects osteoarthritis, focusing on its role in chondrocyte inflammation, apoptosis, and extracellular matrix (ECM) degradation. METHODS: Quantitative real-time PCR was used to measure miR-98-5p and CASP3 mRNA levels in OA cartilage tissues and IL-1ß-treated CHON-001 cells. We predicted miR-98-5p and CASP3 binding sites using TargetScan and confirmed them via luciferase reporter assays. Chondrocyte viability was analyzed using CCK-8 assays, while pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) were quantified via ELISA. Caspase-3 activity was examined to assess apoptosis, and Western blotting was conducted for protein marker quantification. RESULTS: Our results showed lower miR-98-5p levels in both OA cartilage and IL-1ß-stimulated cells. Increasing miR-98-5p resulted in reduced pro-inflammatory cytokines, decreased caspase-3 activity, and improved cell viability. Furthermore, miR-98-5p overexpression hindered IL-1ß-induced ECM degradation, evident from the decline in MMP-13 and ß-catenin levels, and an increase in COL2A1 expression. MiR-98-5p's impact on CASP3 mRNA directly influenced its expression. Mimicking miR-98-5p's effects, CASP3 knockdown also inhibited IL-1ß-induced inflammation, apoptosis, and ECM degradation. In contrast, CASP3 overexpression negated the suppressive effects of miR-98-5p. CONCLUSIONS: In conclusion, our data collectively suggest that miR-98-5p plays a protective role against IL-1ß-induced damage in chondrocytes by targeting CASP3, highlighting its potential as a therapeutic target for OA.

Leukocyte telomere length is associated with MRI-thigh fat-free muscle volume: data from 16 356 UK Biobank adults.

BACKGROUND: Telomere attrition may share common biological mechanisms with bone and muscle loss with aging. Here, we investigated the association between these hallmarks of aging using data from UK Biobank, a large observational study. METHODS: Leukocyte telomere length (LTL as T/S ratio) was measured using a multiplex qPCR assay at baseline (2006-2010). Bone mineral density (whole body and regional; via dual-energy X-ray absorptiometry), trabecular bone score (via lumbar-spine dual-energy X-ray absorptiometry images), fat-free muscle volume (thighs; via magnetic resonance imaging), and muscle fat infiltration (thighs; via magnetic resonance imaging) were measured during the imaging visit (2014-2018). Regression models were used to model LTL against a muscle or bone outcome, unadjusted and adjusted for covariates. RESULTS: A total of 16 356 adults (mean age: 62.8 ± 7.5 years, 50.5% women) were included. In the fully adjusted model, thigh fat-free muscle volume was associated with LTL in the overall sample (adjusted standardized ß (aß) = 0.017, 95% CI 0.009 to 0.026, P < 0.001, per SD increase in LTL), with stronger associations in men (aß = 0.022, 95% CI 0.010 to 0.034, P < 0.001) than in women (aß = 0.013, 95% CI 0.000 to 0.025, P = 0.041) (sex-LTL P = 0.028). The adjusted odds ratio (aOR) for low thigh fat-free muscle volume (body mass index-adjusted, sex-specific bottom 20%) was 0.93 per SD increase in LTL (95% CI 0.89 to 0.96, P < 0.001) in the overall sample, with stronger associations in men (aOR = 0.92, 95% CI 0.87 to 0.99, P = 0.008) than women (aOR = 0.93, 95% CI 0.88 to 0.98, P = 0.009), although the sex difference was not statistically significant in this model (sex-LTL P = 0.37). LTL was not associated with bone mineral density, trabecular bone score, or muscle fat infiltration in the overall or subgroup analyses (P > 0.05). CONCLUSIONS: LTL was consistently associated with thigh fat-free muscle volume in men and women. Future research should investigate moderating effects of lifestyle factors (e.g., physical activity, nutrition, or chronic diseases) in the association between LTL and muscle volume.

Are the tests overpowered or underpowered? A unified solution to correctly specify type I errors in design of clinical trials for two sample proportions.

As one of the most commonly used data types, methods in testing or designing a trial for binary endpoints from two independent populations are still being developed until recently. However, the power and the minimum required sample size comparisons between different tests may not be valid if their type I errors are not controlled at the same level. In this article, we unify all related testing procedures into a decision framework, including both frequentist and Bayesian methods. Sufficient conditions of the type I error attained at the boundary of hypotheses are derived, which help reduce the magnitude of the exact calculations and lay out a foundation for developing computational algorithms to correctly specify the actual type I error. The efficient algorithms are thus proposed to calculate the cutoff value in a deterministic decision rule and the probability value in a randomized decision rule, such that the actual type I error is under but closest to, or equal to, the intended level, respectively. The algorithm may also be used to calculate the sample size to achieve the prespecified type I error and power. The usefulness of the proposed methodology is further demonstrated in the power calculation for designing superiority and noninferiority trials.

Proteomic aging clock (PAC) predicts age-related outcomes in middle-aged and older adults.

Beyond mere prognostication, optimal biomarkers of aging provide insights into qualitative and quantitative features of biological aging and might, therefore, offer useful information for the testing and, ultimately, clinical use of gerotherapeutics. We aimed to develop a proteomic aging clock (PAC) for all-cause mortality risk as a proxy of biological age. Data were from the UK Biobank Pharma Proteomics Project, including 53,021 participants aged between 39 and 70 years and 2,923 plasma proteins assessed using the Olink Explore 3072 assay®. The Spearman correlation between PAC proteomic age and chronological age was 0.77. A total of 10.9% of the participants died during a mean follow-up of 13.3 years, with the mean age at death 70.1 years. We developed a proteomic aging clock (PAC) for all-cause mortality risk as a surrogate of BA using a combination of least absolute shrinkage and selection operator (LASSO) penalized Cox regression and Gompertz proportional hazards models. PAC showed robust age-adjusted associations and predictions for all-cause mortality and the onset of various diseases in general and disease-free participants. The proteins associated with PAC were enriched in several processes related to the hallmarks of biological aging. Our results expand previous findings by showing that age acceleration, based on PAC, strongly predicts all-cause mortality and several incident disease outcomes. Particularly, it facilitates the evaluation of risk for multiple conditions in a disease-free population, thereby, contributing to the prevention of initial diseases, which vary among individuals and may subsequently lead to additional comorbidities.

A comprehensive strategy for the identification of biologics by liquid-chromatography-mass spectrometry for release testing in a regulated environment.

Identification (ID) testing is a regulatory requirement for biopharmaceutical manufacturing, requiring robust, GMP-qualified assays that can distinguish the therapeutic from any other in the facility. Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical tool used to identify and characterize biologics. While routinely leveraged for characterization, LC-MS is relatively rare in Quality Control (QC) settings due to its perceived complexity and scarcity of MS-trained personnel. However, employing LC-MS for identification of drug products has many advantages versus conventional ID techniques, including but not limited to its high specificity, rapid turn-around time, and ease of method execution. In this work, we outline the development and implementation of a comprehensive LC-MS based ID strategy for biologics release testing. Two main workflows (WFs) were developed: i) WF1, a subunit-based assay measuring the molecular weight of the light chain (LC) and heavy chain (HC) of an antibody upon reduction, and ii) WF2, intact mass measurement of the biologic upon N-deglycosylation by PNGase F. The proposed strategy is shown to be applicable for over 40 diverse model biologics including monoclonal antibodies (mAbs), biobetters such as antibody prodrugs/afucosylated mAbs, fusion proteins, multi-specific antibodies, Fabs, and large peptides, all with excellent mass accuracy (error typically < 20 ppm) and precision. It requires a single-step sample preparation and a single click to run and process the data upon method setup. This strategy has been successfully implemented for release testing in GMP labs. Challenges and considerations for the establishment of QC-friendly methods are discussed. It is also shown that these methods can be applied to the ID of more analytically complex biotherapeutics, such as fixed-dose combination (FDC) and drug products co-formulated with trace-level additives.

Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

Country-based rate of emissions reductions should increase by 80% beyond nationally determined contributions to meet the 2 °C target.

The 2015 Paris Agreement aims to keep global warming by 2100 to below 2°C, with 1.5°C as a target. To that end, countries agreed to reduce their emissions by nationally determined contributions (NDCs). Using a fully statistically based probabilistic framework, we find that the probabilities of meeting their nationally determined contributions for the largest emitters are low, e.g. 2% for the USA and 16% for China. On current trends, the probability of staying below 2°C of warming is only 5%, but if all countries meet their nationally determined contributions and continue to reduce emissions at the same rate after 2030, it rises to 26%. If the USA alone does not meet its nationally determined contribution, it declines to 18%. To have an even chance of staying below 2°C, the average rate of decline in emissions would need to increase from the 1% per year needed to meet the nationally determined contributions, to 1.8% per year.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

On-column disulfide bond formation of monoclonal antibodies during Protein A chromatography eliminates low molecular weight species and rescues reduced antibodies.

Disulfide bond reduction, which commonly occurs during monoclonal antibody (mAb) manufacturing processes, can result in a drug substance with high levels of low molecular weight (LMW) species that may fail release specifications because the drug's safety and the efficiency may be affected by the presence of this material. We previously studied disulfide reoxidation of mAbs and demonstrated that disulfide bonds could be reformed from the reduced antibody via redox reactions under an optimal redox condition on Protein A resin. The study here implements a redox system in a manufacturing setting to rescue the reduced mAb product and to further eliminate LMW issues in downstream processing. As such, we incorporate the optimized redox system as one of the wash buffers in Protein A chromatography to enable an on-column disulfide reoxidation to form intact antibody in vitro. Studies at laboratory scale (1 cm (ID) x 20 cm (Height), MabSelect SuRe LX) and pilot scale (30 cm (ID) x 20 cm (Height), MabSelect SuRe LX) were performed to demonstrate the effectiveness and robustness of disulfide formation with multiple mAbs using redox wash on Protein A columns. By applying this rescue strategy using ≤50 g/L-resin loading, the intact mAb purity was improved from <5% in the Protein A column load to >90% in the Protein A column elution with a product yield of >90%. Studies were also done to confirm that adding the redox wash has no negative impact on process yield or impurity removal or product quality. The rescued mAbs were confirmed to form complete interchain disulfide bonds, exhibiting comparable biophysical properties to the reference material. Furthermore, since the redox wash is followed by a bridging buffer wash before the final elution, no additional burden is involved in removing the redox components during the downstream steps. Due to its ease of implementation, significant product purity improvement, and minimal impact on other product quality attributes, we demonstrate that the on-column reoxidation using a redox system is a powerful, simple, and safe tool to recover reduced mAb during manufacturing. Moreover, the apparent benefits of using a high-pH redox wash may further drive the evolution of Protein A platform processes.

ACCOUNTING FOR UNCERTAINTY ABOUT PAST VALUES IN PROBABILISTIC PROJECTIONS OF THE TOTAL FERTILITY RATE FOR MOST COUNTRIES.

Since the 1940s, population projections have in most cases been produced using the deterministic cohort component method. However, in 2015, for the first time, in a major advance, the United Nations issued official probabilistic population projections for all countries based on Bayesian hierarchical models for total fertility and life expectancy. The estimates of these models and the resulting projections are conditional on the UN's official estimates of past values. However, these past values are themselves uncertain, particularly for the majority of the world's countries that do not have longstanding high-quality vital registration systems, when they rely on surveys and censuses with their own biases and measurement errors. This paper extends the UN model for projecting future total fertility rates to take account of uncertainty about past values. This is done by adding an additional level to the hierarchical model to represent the multiple data sources, in each case estimating their bias and measurement error variance. We assess the method by out-of-sample predictive validation. While the prediction intervals produced by the extant method (which does not account for this source of uncertainty) have somewhat less than nominal coverage, we find that our proposed method achieves closer to nominal coverage. The prediction intervals become wider for countries for which the estimates of past total fertility rates rely heavily on surveys rather than on vital registration data, especially in high fertility countries.

Subunit mass analysis for monitoring multiple attributes of monoclonal antibodies.

RATIONALE: Multi-Attribute Methods (MAMs) are appealing due to their ability to provide data on multiple molecular attributes from a single assay. If fully realized, such tests could reduce the number of assays required to support a product control strategy while providing equivalent or greater product understanding relative to the conventional approach. In doing so, MAMs have the potential to decrease development and manufacturing costs by reducing the number of tests in a release panel. METHODS: In this work, we report a MAM which is based on subunit mass analysis. RESULTS: The MAM assay is shown to be suitable for use as a combined method for identity testing, glycan profiling, and protein ratio determination for co-formulated monoclonal antibody (mAb) drugs. This is achieved by taking advantage of the high mass accuracy and relative quantification capabilities of intact mass analysis using quadrupole time-of-flight mass spectrometry (Q-TOF MS). Protein identification is achieved by comparing the measured masses of light chain (LC) and heavy chain (HC) mAbs against their theoretical values. Specificity is based on instrument mass accuracy. Glycan profiling and relative protein ratios are determined by the relative peak intensities of the protein HC glycoforms and LC glycoforms, respectively. Results for these relative quantifications agree well with those obtained by the conventional hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC methods. CONCLUSIONS: The suitability of this MAM for use in a quality control setting is demonstrated through assessment specificity for mAb identity, and accuracy, precision, linearity and robustness for glycan profiling and ratio determination. Results from this study indicate that a MAM with subunit mass analysis has the potential to replace three conventional methods widely used for mAb release testing including identification assay, glycosylation profiling, and ratio determination for co-formulated mAbs.

Study of Guanxinning Injection on Regulatory Mechanism of Bcl-2 and Bax by Liquid Nitrogen Freezing-Mediated Femoral Head Necrosis.

The objective of this study was to investigate the impact of femoral head perfusion by traditional Chinese medicine Guanxinning injection promoting blood circulation for removing blood stasis on the expression of Bcl-2 and Bax induced by liquid nitrogen freezing-mediated femoral head necrosis. 90 rabbits were randomized into three groups. Normal control group was not subjected to any medication. Saline and Guanxinning group were perfused with 0.9% saline and Guanxinning injection once every three days through the hip joint, respectively. Six animals in each group were sacrificed at weeks 1, 3, 6, 9, and 12. PCR and Western blot measured the expressions of Bcl-2 and Bax in the femoral head. The bax expression in the Guanxinning group reduced at the third week significantly compared to the normal control group (P < 0.01). The Bcl-2 expression in the Guanxinning group increased substantially at the third week (P < 0.05 or P < 0.01). Prolonged treatment elevated the expression of Bcl-2 in the Guanxinning group while that of Bax reduced remarkably (P < 0.01). Moreover, the ratio of Bcl-2 to Bax increased gradually in the Guanxinning group with prolonged drug administration. Guanxinning injection can inhibit the cell apoptosis of femoral head necrosis through the treatment by femoral head perfusion.

Less Than 2 °C Warming by 2100 Unlikely.

The recently published Intergovernmental Panel on Climate Change (IPCC) projections to 2100 give likely ranges of global temperature increase in four scenarios for population, economic growth and carbon use1. However these projections are not based on a fully statistical approach. Here we use a country-specific version of Kaya's identity to develop a statistically-based probabilistic forecast of CO2 emissions and temperature change to 2100. Using data for 1960-2010, including the UN's probabilistic population projections for all countries2-4, we develop a joint Bayesian hierarchical model for GDP per capita and carbon intensity. We find that the 90% interval for cumulative CO2 emissions includes the IPCC's two middle scenarios but not the extreme ones. The likely range of global temperature increase is 2.0-4.9°C, with median 3.2°C and a 5% (1%) chance that it will be less than 2°C (1.5°C). Population growth is not a major contributing factor. Our model is not a "business as usual" scenario, but rather is based on data which already show the effect of emission mitigation policies. Achieving the goal of less than 1.5°C warming will require carbon intensity to decline much faster than in the recent past.

Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis.

In the phenylpropanoid production process, p-coumaric acid is the most important intermediate metabolite. It is generally accepted that the activity of tyrosine ammonia-lyase (TAL), which converts L-tyrosine to p-coumaric acid, represents the rate-limiting step. Therefore, an error-prone PCR-based random mutagenesis strategy was utilized for screening variants with higher catalytic activity. After rounds of screening, three variant enzymes were obtained, exhibiting improved production rates of 41.2, 37.1, and 38.0 %, respectively. Variants associated with increased expression level (S9N), improved catalytic efficiency (A11T), and enhanced affinity between TAL and L-tyrosine (E518V) were identified as beneficial amino acid substitutions by site-directed mutagenesis. Combining all of the beneficial amino acid substitutions, a variant, MT-S9N/-A11T/-E518V, exhibiting the highest catalytic activity was obtained. The K m value of MT-S9N/-A11T/-E518V decreased by 25.4 % compare to that of wild-type, while the activity, k cat/K m, and p-coumaric-acid yield were improved by 36.5, 31.2, and 65.9 %, respectively. Furthermore, the secondary structure of the 5'-end of MT-S9N mRNA and the three-dimensional protein structure of MT-E518V were modeled. Therefore, two potential mechanisms were speculated: (1) a simplified mRNA 5'-end secondary structure promotes TAL expression and (2) anchoring the flexible loop region (Glu325-Arg336) to maintain the active-site pocket opening ensures easy access by the L-tyrosine to the active site and thus improves p-coumaric acid yields.

Identification and application of keto acids transporters in Yarrowia lipolytica.

Production of organic acids by microorganisms is of great importance for obtaining building-block chemicals from sustainable biomass. Extracellular accumulation of organic acids involved a series of transporters, which play important roles in the accumulation of specific organic acid while lack of systematic demonstration in eukaryotic microorganisms. To circumvent accumulation of by-product, efforts have being orchestrated to carboxylate transport mechanism for potential clue in Yarrowia lipolytica WSH-Z06. Six endogenous putative transporter genes, YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470g, α-ketoglutarate accumulated to 46.7 g·L(-1), whereas the concentration of pyruvate decreased to 12.3 g·L(-1). Systematic identification of these keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms.

Identification of membrane proteins associated with phenylpropanoid tolerance and transport in Escherichia coli BL21.

Phenylpropanoids are phytochemicals produced by some plants and possess a wide variety of biological activities. These compounds exist in plants in low amounts. Production of them in genetically engineered microorganisms has many advantages. A majority of functional phenylpropanoids are toxic to microbial hosts. Export of these compounds may relieve the cellular toxicity and increase the yield. However, proteins and mechanisms involved in phenylpropanoids transport and tolerance remain poorly understood. In this study, 16 membrane proteins that were differentially expressed in Escherichia coli in response to three typical phenylpropanoids (resveratrol, naringenin and rutin) were identified using a membrane proteomics approach. These proteins included outer membrane proteins OmpA, OmpF, OmpW, FadL, TolC, LamB, and YaeT, peripheral membrane proteins AtpD, AtpH, YgaU, OppA, MalK, and MalE, and cytoplasmic membrane proteins OppD, PotG, and ManX. Functions of these proteins were determined by using gene overexpression and silencing. The results suggest that OmpA and FadL may play important roles in the transmembrane export of phenylpropanoids in E. coli. LamB, MalE, MalK and ManX may participate in phenylpropanoid uptake. The role of YgaU in enhancing the tolerance to phenylpropanoids remains to be determined. These results may assist the engineering of microorganisms with enhanced phenylpropanoid producing capabilities. BIOLOGICAL SIGNIFICANCE: Phenylpropanoids are phytochemicals produced by some plants and possess a wide variety of biological activities. Both the tolerance and the transport of phenylpropanoids play important roles in systematic metabolic engineering of microorganisms to produce these phytochemicals. Both specific and non-specific transporters are essential for these functions but remain poorly understood. This research utilized membrane proteomics to identify E. coli BL21 (DE3) membrane proteins that may be involved in phenylpropanoid transport and tolerance. These results may facilitate the construction of more efficient microbial phenylpropanoid producers through genetic engineering of membrane transporter proteins.

Multivariate modular metabolic engineering of Escherichia coli to produce resveratrol from L-tyrosine.

Microbial fermentations and bioconversion promise to revolutionize the conventional extraction of resveratrol from natural plant sources. However, the development of efficient and feasible microbial processes remains challenging. Current fermentation strategies often require supplementation of expensive phenylpropanoic precursors and two separate fermentation protocols, which are significantly more difficult and expensive to undertake when migrating to large-scale fermentation processes. In this study, an Escherichia coli fermentation system, consisting of tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), stilbene synthase (STS), malonate synthetase, and malonate carrier protein, was developed to produce resveratrol from L-tyrosine. Multivariate modular metabolic engineering, which redefined the overall pathway as a collection of distinct modules, was employed to assess and alleviate pathway bottlenecks. Using this strategy, the optimum strain was capable of producing 35.02 mg/L of resveratrol from L-tyrosine in a single medium. The strategy described here paves the way to the development of a simple and economical process for microbial production of resveratrol and other similar stilbene chemicals.

Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping.

Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (∼0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.

A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins.

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.