Mass spectrometry (MS)-based quantification of highly homologous proteins in complex samples has proven difficult due to subtle sequence variations and the wide dynamic range of protein isoforms present. Herein, we report the use of reductive dimethylation on intact proteins to quantitatively compare protein isoform expression in the nucleus and cytoplasm of mesenchymal stem cells (MSC) and normal stroma. By coupling fixed-charge MS/MS scanning, high-resolution UPLC FT-MS data-dependent acquisition and MASCOT-based data mining, hydrogen/deuterium-labeled dimethyl-lysine peptides were simultaneously captured allowing the accurate comparison of 123 protein isoforms in parallel LC MS/MS runs. Thirty-four isoforms were identified that had expression levels specific to MSC. Where possible, proteomic analyses were verified by Western blotting and were demonstrated to be divergent from the level of gene transcription detected for certain proteins. Our analysis provides a protein isoform signature specific to MSC and demonstrates the suitability of dimethyl-lysine labeling on intact proteins for quantifying highly homologous proteins on a proteome-wide scale.

Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105-) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.

The outbreak of a pandemic influenza H1N1 in 2009 required the rapid generation of high-yielding vaccines against the A/California/7/2009 virus, which were achieved by either addition or deletion of a glycosylation site in the influenza proteins hemagglutinin and neuraminidase. In this report, we have systematically evaluated the glycan composition, structural distribution and topology of glycosylation for two high-yield candidate reassortant vaccines (NIBRG-121xp and NYMC-X181A) by combining various enzymatic digestions with high performance liquid chromatography and multiple-stage mass spectrometry. Proteomic data analyses of the full-length protein sequences determined 9 N-glycosylation sites of hemagglutinin, and defined 6 N-glycosylation sites and the glycan structures of low abundance neuraminidase, which were occupied by high-mannose, hybrid and complex-type N-glycans. A total of ~300 glycopeptides were analyzed and manually validated by tandem mass spectrometry. The specific N-glycan structure and topological location of these N-glycans are highly correlated to the spatial protein structure and the residential ligand binding. Interestingly, sulfation, fucosylation and bisecting N-acetylglucosamine of N-glycans were also reliably identified at the specific glycosylation sites of the two influenza proteins that may serve a crucial role in regulating the protein structure and increasing the protein abundance of the influenza virus reassortants.