The application of HPLC/MS analysis with a multi-enzyme digest strategy to characterize different interferon product variants produced from Pichia pastoris.

Interferons are signaling proteins that belong to the large class of cytokines and human interferons which are classified based on the type of receptor interactions: type I, II and III. IFNα2b belongs to the type I interferon class with a major therapeutic application for the treatment of hepatitis B and C infections. A recombinant form of IFNα2b expressed in E.

On-demand manufacturing of clinical-quality biopharmaceuticals.

Conventional manufacturing of protein biopharmaceuticals in centralized, large-scale, single-product facilities is not well-suited to the agile production of drugs for small patient populations or individuals. Previous solutions for small-scale manufacturing are limited in both process reproducibility and product quality, owing to their complicated means of protein expression and purification. We describe an automated, benchtop, multiproduct manufacturing system, called Integrated Scalable Cyto-Technology (InSCyT), for the end-to-end production of hundreds to thousands of doses of clinical-quality protein biologics in about 3 d.

Integrated Bottom-Up and Top-Down Liquid Chromatography-Mass Spectrometry for Characterization of Recombinant Human Growth Hormone Degradation Products.

With the advent of biosimilars to the U.S. market, it is important to have better analytical tools to ensure product quality from batch to batch.

Advances in the Chromosome-Centric Human Proteome Project: looking to the future.

The mission of the Chromosome-Centric Human Proteome Project (C-HPP), is to map and annotate the entire predicted human protein set (~20,000 proteins) encoded by each chromosome. The initial steps of the project are focused on 'missing proteins (MPs)', which lacked documented evidence for existence at protein level. In addition to remaining 2,579 MPs, we also target those annotated proteins having unknown functions, uPE1 proteins, alternative splice isoforms and post-translational modifications.

Characterization of Site-Specific Glycosylation in Influenza A Virus Hemagglutinin Produced by Spodoptera frugiperda Insect Cell Line.

Influenza hemagglutinin is a surface glycoprotein related to virus invasion and host immune system response. Understanding site specific glycosylation of hemagglutinin will increase our knowledge about virus evolution and can improve the design and quality of vaccines. In our study, we used glycoproteomic analysis based on multienzyme digestion followed by LC tandem MS analysis to determine the glycosylation of Influenza hemagglutinin (H1/A/California/04/2009) using the following steps: PNGaseF treatment combined with trypsin or pepsin digestion was used to determine the glycosites and glycan occupancy.

Integrated Proteomic and Transcriptomic-Based Approaches to Identifying Signature Biomarkers and Pathways for Elucidation of Daoy and UW228 Subtypes.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Patient survival has remained largely the same for the past 20 years, with therapies causing significant health, cognitive, behavioral and developmental complications for those who survive the tumor. In this study, we profiled the total transcriptome and proteome of two established MB cell lines, Daoy and UW228, using high-throughput RNA sequencing (RNA-Seq) and label-free nano-LC-MS/MS-based quantitative proteomics, coupled with advanced pathway analysis.

Human Proteome Project Mass Spectrometry Data Interpretation Guidelines 2.1.

Every data-rich community research effort requires a clear plan for ensuring the quality of the data interpretation and comparability of analyses. To address this need within the Human Proteome Project (HPP) of the Human Proteome Organization (HUPO), we have developed through broad consultation a set of mass spectrometry data interpretation guidelines that should be applied to all HPP data contributions. For submission of manuscripts reporting HPP protein identification results, the guidelines are presented as a one-page checklist containing 15 essential points followed by two pages of expanded description of each.

Identifying N-Glycan Biomarkers in Colorectal Cancer by Mass Spectrometry.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. Delineating biological markers (biomarkers) for early detection, when treatment is most effective, is key to prevention and long-term survival of patients. Development of reliable biomarkers requires an increased understanding of the CRC biology and the underlying molecular and cellular mechanisms of the disease.

Microfluidic Platform for Assessment of Therapeutic Proteins Using Molecular Charge Modulation Enhanced Electrokinetic Concentration Assays.

Therapeutic proteins (TPs) are critical in modern medicine, yet shortage of TPs in disaster situations and remote areas remains a worldwide challenge. Manufacturing and real-time release of TPs on demand at the point-of-care is considered the key to this issue, which requires reliable and rapid analytics techniques for quality assurance. Herein we report a microfluidic platform that could be implemented in-line and at the point-of-care for real-time decision-making about the quality of a TP.

Quantitative Profiling Identifies Potential Regulatory Proteins Involved in Development from Dauer Stage to L4 Stage in Caenorhabditis elegans.

When Caenorhabditis elegans encounters unfavorable growth conditions, it enters the dauer stage, an alternative L3 developmental period. A dauer larva resumes larval development to the normal L4 stage by uncharacterized postdauer reprogramming (PDR) when growth conditions become more favorable. During this transition period, certain heterochronic genes involved in controlling the proper sequence of developmental events are known to act, with their mutations suppressing the Muv (multivulva) phenotype in C.

Systems Proteomics View of the Endogenous Human Claudin Protein Family.

Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein-protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity.

Integrated Proteomic and Genomic Analysis of Gastric Cancer Patient Tissues.

V-erb-b2 erythroblastic leukemia viral oncogene homologue 2, known as ERBB2, is an important oncogene in the development of certain cancers. It can form a heterodimer with other epidermal growth factor receptor family members and activate kinase-mediated downstream signaling pathways. ERBB2 gene is located on chromosome 17 and is amplified in a subset of cancers, such as breast, gastric, and colon cancer.

Combination of Multiple Spectral Libraries Improves the Current Search Methods Used to Identify Missing Proteins in the Chromosome-Centric Human Proteome Project.

Approximately 2.9 billion long base-pair human reference genome sequences are known to encode some 20 000 representative proteins. However, 3000 proteins, that is, ~15% of all proteins, have no or very weak proteomic evidence and are still missing.

GenomewidePDB 2.0: A Newly Upgraded Versatile Proteogenomic Database for the Chromosome-Centric Human Proteome Project.

Since the launch of the Chromosome-centric Human Proteome Project (C-HPP) in 2012, the number of "missing" proteins has fallen to 2932, down from ∼5932 since the number was first counted in 2011. We compared the characteristics of missing proteins with those of already annotated proteins with respect to transcriptional expression pattern and the time periods in which newly identified proteins were annotated. We learned that missing proteins commonly exhibit lower levels of transcriptional expression and less tissue-specific expression compared with already annotated proteins.

In-depth N-glycome profiling of paired colorectal cancer and non-tumorigenic tissues reveals cancer-, stage- and EGFR-specific protein N-glycosylation.

Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-liquid chromatography-tandem mass spectrometry-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-Glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.

Quest for Missing Proteins: Update 2015 on Chromosome-Centric Human Proteome Project.

This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed "missing proteins") in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP's activities; therefore, we have summarized some of the key approaches and their roles in the project.

Quantitative proteomic analysis of paired colorectal cancer and non-tumorigenic tissues reveals signature proteins and perturbed pathways involved in CRC progression and metastasis.

Modern proteomics has proven instrumental in our understanding of the molecular deregulations associated with the development and progression of cancer. Herein, we profile membrane-enriched proteome of tumor and adjacent normal tissues from eight CRC patients using label-free nanoLC-MS/MS-based quantitative proteomics and advanced pathway analysis. Of the 948 identified proteins, 184 proteins were differentially expressed (P<0.

Clusterin glycopeptide variant characterization reveals significant site-specific glycan changes in the plasma of clear cell renal cell carcinoma.

Cancer-related alterations in protein glycosylation may serve as diagnostic or prognostic biomarkers or may be used for monitoring disease progression. Clusterin is a medium abundance, yet heavily glycosylated, glycoprotein that is upregulated in clear cell renal cell carcinoma (ccRCC) tumors. We recently reported that the N-glycan profile of clusterin is altered in the plasma of ccRCC patients.

Development of an improved fractionation of the human plasma proteome by a combination of abundant proteins depletion and multi-lectin affinity chromatography.

Aim: Current analytical tools lack the required capacity to reduce the complexity of the plasma proteome and identify low-level proteins of clinical interest. Hence, the need to develop a fractionation approach to provide adequate throughput for a clinical study and minimize the loss and improve the detection of low abundance proteins.

Materials & Methods: We present the development of an analytical platform that combines the depletion of 12 high abundance proteins and multi-lectin affinity chromatography (12P-M-LAC) fractionation.

Use of ENCODE resources to characterize novel proteoforms and missing proteins in the human proteome.

We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the "missing proteins", novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques.

Identification of novel glycans with disialylated structures in α3 integrin from mouse kidney cells with the phenotype of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder caused by mutations in the Pkd1 or Pkd2 genes, in which large cysts replace normal kidney tissue, leading to end-stage kidney disease. In this study we have utilized a powerful nano-HPLC-mass spectrometric approach to characterize patterns of normal and abnormal N-linked glycosylation of α3 integrin subunit in Pkd1(-/-) cells derived from mouse kidneys. Higher molecular weight glycan structures with a different monosaccharide composition were observed at two sites, namely, Asn-925 and Asn-928 sites in α3 integrin isolated from Pkd1(+/+) cells compared with Pkd1(-/-) cells.

Comprehensive N-glycome profiling of cultured human epithelial breast cells identifies unique secretome N-glycosylation signatures enabling tumorigenic subtype classification.

The secreted cellular sub-proteome (secretome) is a rich source of biologically active glycoproteins. N-Glycan profiling of secretomes of cultured cancer cells provides an opportunity to investigate the link between protein N-glycosylation and tumorigenesis. Utilizing carbon-LC-ESI-CID-MS/MS of protein released native N-glycans, we accurately profiled the secretome N-glycosylation of six human epithelial breast cells including normal mammary epithelial cells (HMEC) and breast cancer cells belonging to luminal A subtype (MCF7), HER2-overexpressing subtype (SKBR3), and basal B subtype (MDA-MB157, MDA-MB231, HS578T).