Amino Acid Analysis by Means of MALDI TOF Mass Spectrometry or MALDI TOF/TOF Tandem Mass Spectrometry.

Here we describe two different AAA protocols based on application of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS). First protocol describes a MALDI TOF MS-based method for a routine simultaneous qualitative and quantitative analysis of free amino acids and protein hydrolysates. Linear responses between the amino acid concentration and the peak intensity ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 μM.

Impact of Influenza A Virus Infection on the Proteomes of Human Bronchoepithelial Cells from Different Donors.

Susceptibility to influenza A virus is determined by a balance of viral and host factors. The genetic background of the host contributes to the severity of disease, but the influenza-related proteomes of cells from different individuals have not been compared. We used high-resolution mass spectrometry to identify proteins in normal human bronchial epithelial (NHBE) cells isolated from three different donors.

The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging.

Bone-marrow derived mesenchymal stromal cells (BMSCs) have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5], [6].

System-wide survey of proteomic responses of human bone marrow stromal cells (hBMSCs) to in vitro cultivation.

Human bone marrow stromal cells (hBMSCs, also loosely called bone marrow-derived mesenchymal stem cells) are the subject of increasing numbers of clinical trials and laboratory research. Our group recently reported on the optimization of a workflow for a sensitive proteomic study of hBMSCs. Here, we couple this workflow with a label-free protein quantitation method to investigate the molecular responses of hBMSCs to long-term in vitro passaging.

Development of a protein marker panel for characterization of human induced pluripotent stem cells (hiPSCs) using global quantitative proteome analysis.

The emergence of new methods for reprogramming of adult somatic cells into induced pluripotent stem cells (iPSC) led to the development of new approaches in drug discovery and regenerative medicine. Investigation of the molecular mechanisms underlying the self-renewal, expansion and differentiation of human iPSC (hiPSC) should lead to improvements in the manufacture of safe and reliable cell therapy products. The goal of our study was qualitative and quantitative proteomic characterizations of hiPSC by means of electrospray ionization (ESI)-MS(e) and MALDI-TOF/TOF mass spectrometry (MS).

Stability of neuraminidase in inactivated influenza vaccines.

Influenza vaccines are effective in protecting against illness and death caused by this seasonal pathogen. Antibodies that block the function of either hemagglutinin (HA) or neuraminidase (NA) contribute to vaccine efficacy, however vaccine potency is based only on HA content. NA protein content in vaccines varies from season to season due to differences in the relative amounts of HA and NA in influenza A, H1N1 and H3N2, and influenza B viruses that are selected for each manufacturing campaign.

Global proteomic signature of undifferentiated human bone marrow stromal cells: evidence for donor-to-donor proteome heterogeneity.

The clinical application of human bone marrow stromal cells (hBMSCs) largely depends on their capacity to expand in vitro. We have conducted a comprehensive comparative proteomic analysis of culture-expanded hBMSCs obtained from different human donors. The data reveal extensive donor-to-donor proteomic heterogeneity.

Potency under pressure: the impact of hydrostatic pressure on antigenic properties of influenza virus hemagglutinin.

Background: Influenza vaccines are effective in protecting against illness and death caused by this seasonal pathogen. The potency of influenza vaccines is measured by single radial immunodiffusion (SRID) assay that quantifies antigenic forms of hemagglutinin (HA). Hydrostatic pressure results in loss of binding of influenza virus to red blood cells, but it is not known whether this infers loss of potency.

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450.

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data.

Improved proteomic profiling of the cell surface of culture-expanded human bone marrow multipotent stromal cells.

A comprehensive analysis of the membrane proteome is essential to explain the biology of multipotent stromal cells and identify reliable protein biomarkers for the isolation as well as tracking of cells during differentiation and maturation. However, proteomic analysis of membrane proteins is challenging and they are noticeably under-represented in numerous proteomic studies. Here we introduce new approach, which includes high pressure-assisted membrane protein extraction, protein fractionation by gel-eluted liquid fraction entrapment electrophoresis (GELFREE), and combined use of liquid chromatography MALDI and ESI tandem mass spectrometry.

Label-free mass spectrometry-based quantification of hemagglutinin and neuraminidase in influenza virus preparations and vaccines.

Background: Influenza vaccination is the primary method for preventing influenza and its severe complications. An accurate rapid method to determine hemagglutinin (HA) concentration would facilitate reference antigen preparation and consequently expedite availability of seasonal as well as pandemic vaccines.

Objective: The goal of this study was to develop a label-free mass spectrometry (MS) based method that enables simultaneous identification and quantification of HA, neuraminidase (NA), and other viral proteins and protein contaminations in influenza vaccine or virus preparations.

Amino acid analysis by means of MALDI TOF mass spectrometry or MALDI TOF/TOF tandem mass spectrometry.

Here, we describe two different amino acid analysis protocols based on the application of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS). First protocol describes a MALDI TOF MS-based method for a routine simultaneous qualitative and quantitative analysis of free amino acids and protein hydrolysates (Alterman et al. Anal Biochem 335: 184-191, 2004).

Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis.

Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF)-based label-free relative protein quantification strategy that involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins followed by in-gel trypsin digestion. The main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT).

Protein-based multiplex assays: mock presubmissions to the US Food and Drug Administration.

As a part of ongoing efforts of the NCI-FDA Interagency Oncology Task Force subcommittee on molecular diagnostics, members of the Clinical Proteomic Technology Assessment for Cancer program of the National Cancer Institute have submitted 2 protein-based multiplex assay descriptions to the Office of In Vitro Diagnostic Device Evaluation and Safety, US Food and Drug Administration. The objective was to evaluate the analytical measurement criteria and studies needed to validate protein-based multiplex assays. Each submission described a different protein-based platform: a multiplex immunoaffinity mass spectrometry platform for protein quantification, and an immunological array platform quantifying glycoprotein isoforms.

Proteomics-based characterization of hemagglutinins in different strains of influenza virus.

Infection with influenza A (subtypes H1N1 and H3N2) or B viruses results in over half a million deaths worldwide every year. Frequent antigenic changes (drift) in two major viral surface proteins hemagglutinin (HA) and neuraminidase lead to the constant emergence of antigenically distinct virus strains against which there is sub-optimal immunity in the population. Consequently the suitability of the viral strains included in the trivalent influenza vaccine (TIV) has to be re-evaluated annually.

Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach.

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years.

Utility of polyclonal antibodies targeted toward unique tryptic peptides in the proteomic analysis of cytochrome P450 isozymes.

P450s are key enzymes responsible for biotransformation of numerous endogenous and exogenous compounds and are located in almost every tissue. This superfamily is the largest group of enzymes (>6000) that share a high degree of similarity in protein sequence. The human genome contains 57 CYP genes and 58 pseudogenes.

A proteomic analysis of bromobenzene reactive metabolite targets in rat liver cytosol in vivo.

Metabolic activation and protein covalent binding are early and apparently obligatory events in the cytotoxicity of many simple organic chemicals including drugs and natural products. Although much has been learned about the chemistry of reactive metabolite formation and reactivity toward protein nucleophiles, progress in identifying specific protein targets for reactive metabolites of various protoxins has been much slower. We previously reported nine microsomal and three cytosolic proteins as targets for reactive metabolites of bromobenzene in rat liver.

MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis.

This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 alpha-amino acids, beta-amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.

Protein interactions with subcutaneously implanted biosensors.

Biofouling of in vivo glucose sensors has been indicated as the primary reason for sensitivity losses observed during the first 24 h after implant [Wisniewski N, Moussy F, Reichert WM. Characterization of implantable biosensor membrane biofouling. Fresen J Anal Chem 2000; 366(6-7): 611-621].

Quantitative analysis of cytochrome p450 isozymes by means of unique isozyme-specific tryptic peptides: a proteomic approach.

A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging.

Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1.

Addition of exogenous ceramide causes a significant displacement of cholesterol in lipid raft model membranes. However, whether ceramide-induced cholesterol displacement is sufficient to alter the protein composition of caveolin-enriched lipid raft membranes is unknown. Therefore, we examined whether increasing endogenous ceramide levels with bacterial sphingomyelinase (bSMase) depleted cholesterol and changed the protein composition of caveolin-enriched membranes (CEMs) isolated from immortalized Schwann cells.

Purification and physical-chemical properties of methanobactin: a chalkophore from Methylosinus trichosporium OB3b.

Methanobactin is an extracellular, copper-binding chromopeptide from the methane-oxidizing bacterium, Methylosinus trichosporium OB3b, believed to be involved in copper detoxification, sequestration, and uptake. Although small (1217.2 Da), methanobactin possesses a complex three-dimensional macrocyclic structure with several unusual moieties.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based amino acid analysis.

Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids.

Methanobactin, a copper-acquisition compound from methane-oxidizing bacteria.

Siderophores are extracellular iron-binding compounds that mediate iron transport into many cells. We present evidence of analogous molecules for copper transport from methane-oxidizing bacteria, represented here by a small fluorescent chromopeptide (C45N12O14H62Cu, 1216 daltons) produced by Methylosinus trichosporium OB3b. The crystal structure of this compound, methanobactin, was resolved to 1.