Biomedical subjects

John T Stults

Publications and source records attributed to John T Stults.

7 recordsLinked to original sources

PubMed2005

Mass spectrometric contributions to the practice of phosphorylation site mapping through 2003: a literature review.

Reversible phosphorylation of proteins is among the most important post-translational modifications, and elucidation of sites of phosphorylation is essential to understanding the regulation of key cellular processes such as signal transduction. Unfortunately phosphorylation site mapping is as technically challenging as it is important. Limitations in the traditional method of Edman degradation of (32)P-labeled phosphoproteins have spurred the development of mass spectrometric methods for phosphopeptide identification and sequencing. To assess the practical contributions of the various technologies we conducted a literature search of publications using mass spectrometry to discover previously unknown phosphorylation sites. 1281 such phosphorylation sites were reported in 203 publications between 1992 and 2003. This review examines and catalogs those methods, identifies the trends that have emerged in the past decade, and presents representative examples from among these methods.

Animals↗

PubMed2005

An automated, sheathless capillary electrophoresis-mass spectrometry platform for discovery of biomarkers in human serum.

A capillary electrophoresis-mass spectrometry (CE-MS) method has been developed to perform routine, automated analysis of low-molecular-weight peptides in human serum. The method incorporates transient isotachophoresis for in-line preconcentration and a sheathless electrospray interface. To evaluate the performance of the method and demonstrate the utility of the approach, an experiment was designed in which peptides were added to sera from individuals at each of two different concentrations, artificially creating two groups of samples. The CE-MS data from the serum samples were divided into separate training and test sets. A pattern-recognition/feature-selection algorithm based on support vector machines was used to select the mass-to-charge (m/z) values from the training set data that distinguished the two groups of samples from each other. The added peptides were identified correctly as the distinguishing features, and pattern recognition based on these peptides was used to assign each sample in the independent test set to its respective group. A twofold difference in peptide concentration could be detected with statistical significance (p-value < 0.0001). The accuracy of the assignment was 95%, demonstrating the utility of this technique for the discovery of patterns of biomarkers in serum.

Automation↗

PubMed2005

Proteomics.

Proteomics is the measurement of one or more protein populations or proteomes, preferably in a quantitative manner. A protein population may be the set of proteins found in an organism, in a tissue or biofluid, in a cell, or in a subcellular compartment. A population also may be the set of proteins with a common characteristic, for example, those that interact with each other in molecular complexes, those involved in the same process such as signal transduction or cell cycle control, or those that share a common posttranslational modification such as phosphorylation or glycosylation. Proteomics experiments that involve mass spectrometry are divided into five categories: (1) protein identification, (2) protein quantitation or differential analysis, (3) protein-protein interactions, (4) post-translational modifications, and (5) structural proteomics. Each of these proteomics categories is reviewed. Examples are given for quantitative experiments involving two-dimensional gel electrophoresis, and for gel-free analysis using isotope-coded affinity tags. The impact of proteomics on biological research and on drug development is discussed. Challenges for further development in proteomics are presented, including sample preparation, sensitivity, dynamic range, and automation.

Animals↗

PubMed2004

Structure-activity relationships by mass spectrometry: identification of novel MMP-3 inhibitors.

A novel class of nonpeptide inhibitors of stromelysin (MMP-3) has been discovered with the use of mass spectrometry. The method relies on the development of structure-activity relationships by mass spectrometry (SAR by MS) and utilizes information derived from the binding of known inhibitors to identify novel inhibitors of a target protein with a minimum of synthetic effort. Noncovalent complexes of known inhibitors with a target protein are analyzed; these inhibitors are deconstructed into sets of fragments which compete for common or overlapping binding sites on the target protein. The binding of each fragment set can be studied independently. With the use of competition studies, novel members of each fragment set are identified from compound libraries that bind to the same site on the target protein. A novel inhibitor of the target protein was then constructed by chemically linking a combination of members of each fragment set in a manner guided by the proximity and orientation of the fragments derived from the known inhibitors. In the case of stromelysin, a novel inhibitor composed of favorably linked fragments was observed to form a 1:1 complex with stromelysin. Compounds that were not linked appropriately formed higher order complexes with stoichiometries of 2:1 or greater. These linked molecules were subsequently assessed for their ability to block stromelysin function in a chromogenic substrate assay.

Mass Spectrometry↗

PubMed2003

Protein identification: the origins of peptide mass fingerprinting.

Peptide mass fingerprinting (PMF) grew from a need for a faster, more efficient method to identify frequently observed proteins in electrophoresis gels. We describe the genesis of the idea in 1989, and show the first demonstration with fast atom bombardment mass spectrometry. Despite its promise, the method was seldom used until 1992, with the coming of significantly more sensitive commercial instrumentation based on MALDI-TOF-MS. We recount the evolution of the method and its dependence on a number of technical breakthroughs, both in mass spectrometry and in other areas. We show how it laid the foundation for high-throughput, high-sensitivity methods of protein analysis, now known as proteomics. We conclude with recommendations for further improvements, and speculation of the role of PMF in the future.

Amino Acid Sequence↗

PubMed2002

Selective detection of membrane proteins without antibodies: a mass spectrometric version of the Western blot.

A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.

Amino Acid Sequence↗

PubMedDate not supplied

Proteomic profiling of surface proteins on Th1 and Th2 cells.

We utilized mass spectrometry to profile cell surface protein differential expression on primary human T helper (Th1 and Th2) cells with the stable isotope labeling by amino acids in cell culture (SILAC) approach. Proteomic and microarray analyses were done concurrently and results were compared for 38 different genes. Although microarray studies displayed wide variability between donors for mRNA expression, these two approaches were shown to be corroborative for most gene products with the exception of a small subset of uncorrelated protein and message levels. The greatest differing Th1 to Th2 ratios were observed for BST2 (bone marrow stromal protein 2) and TRIM (T cell receptor interacting molecule). Both showed greater Th1 expression by proteomic methods, even though mRNA levels were approximately equal for both. To validate this method, we compared protein expression levels of a recently cloned molecule, B and T cell lymphocyte attenuator (BTLA), on Th1 and Th2 cell populations and showed greater protein expression on Th1 cells, which agrees with a previous analysis of higher BTLA mRNA expression in Th1 cells.(1).

Cell Differentiation↗