Biomedical subjects

Mary Ann Gawinowicz

Publications and source records attributed to Mary Ann Gawinowicz.

4 recordsLinked to original sources

PubMed2006

Identification and characterization of two mature isoforms of retinoschisin in murine retina.

Retinoschisin (RS) is a 24 kDa secreted protein expressed in retina and is required for the structural and functional integrity of the retina. RS has been predicted to serve as an adhesive protein but the precise molecular mechanism by which it functions in retina is not yet known. During investigations on structural and functional aspects of RS in murine retina using proteomic tools, we identified two isoforms of RS that differed in mass by 200 Da with no apparent change in charge. Mass spectra and amino acid sequence analysis of the tryptic peptides revealed that these isoforms differed by two amino acids at the N-terminus which suggested processing of RS signal sequence at two cleavage sites by signal peptidase as the basic mechanism underlying the occurrence of two mature RS isoforms in retina. Bioinformatic analysis identified two potential cleavage sites (between amino acids 21-22 and 23-24) in RS signal sequence. The flexibility of the signal peptidase to cleave at two sites is correlated to the amino acid composition of the RS signal sequence. This finding represents a rare example of a naturally occurring signal sequence cleavage at more than one site in vivo.

Amino Acid Sequence↗

PubMed2004

Identification of cytoskeletal [14C]carboplatin-binding proteins reveals reduced expression and disorganization of actin and filamin in cisplatin-resistant cell lines.

Cisplatin resistant (CP-r) cells often show decreased uptake of cisplatin in association with reduced cell surface proteins and decreased endocytosis. In this report, two major [14C]carboplatin-binding proteins were identified as filamin and actin by photoaffinity labeling and mass spectrometry. Decreased expression of these two proteins was found in two different human CP-r cell lines (KB-CP20 and 7404-CP20), in comparison with their parental cell lines (KB-3-1 and BEL-7404), respectively. Disorganization of beta-actin and filamin 250 and 90 was also detected in these CP-r cells by confocal microscopy. Transfection of a wild-type actin-enhanced green fluorescent protein (EGFP) expression vector into 7404-CP20 cells resulted in a nonfilamentous actin-EGFP distribution compared with a normal distribution in the cisplatin-sensitive BEL-7404 cells, suggesting that cytoskeletal organization is disturbed in the CP-r cells. The identification of actin and filamin as [14C]carboplatin-binding proteins and decreased expression and disorganization of several cytoskeletal proteins in CP-r cells provide a molecular and cellular basis for the known defect in endocytosis in these cells.

Actins↗

PubMed2003

Reactivity of functional groups on the protein surface: development of epoxide probes for protein labeling.

We present the development of new affinity probes for protein labeling based on an epoxide reactive group. Systematic screening revealed that an epoxide functionality possesses the special combination of stability and reactivity which renders it stable toward proteins in solution but reactive on the protein surface outside the active site (proximity-induced reactivity). Highly efficient and selective labeling of purified HCA II (human carbonic anhydrase II) was achieved. For instance, 2 equiv of epoxide probe 9 was sufficient for nearly quantitative labeling of HCA II (>90% yield, 20 h reaction time). MS analysis of the labeled protein revealed that 1 equiv of the probe was attached and that labeling occurred at a single residue (His 64) outside the active site. Importantly, epoxide probe 9 selectively labeled HCA II both in simple protein mixtures and in cellular extracts. In addition to the chemical insight and its relevance to many epoxide-containing natural products, this study generated a promising lead in the development of new affinity probes for protein labeling.

Affinity Labels↗

PubMed2003

ABRF-PRG03: phosphorylation site determination.

A fundamental aspect of proteomics is the analysis of post-translational modifications, of which phosphorylation is an important class. Numerous nonradioactivity-based methods have been described for high-sensitivity phosphorylation site mapping. The ABRF Proteomics Research Group has conducted a study to help determine how many laboratories are equipped to take on such projects, which methods they choose to apply, and how successful the laboratories are in implementing particular methodologies. The ABRF-PRG03 sample was distributed as a tryptic digest of a mixture of two proteins with two synthetic phosphopeptides added. Each sample contained 5 pmol of unphosphorylated protein digest, 1 pmol of each phosphopeptide from the same protein, and 200 fmol of a minor protein component. Study participants were challenged to identify the two proteins and the two phosphorylated peptides, and determine the site of phosphorylation in each peptide. Almost all respondents successfully identified the major protein component, whereas only 10% identified the minor protein component. Phosphorylation site analysis proved surprisingly difficult, with only 3 of the 54 laboratories correctly determining both sites of phosphorylation. Various strategies and instruments were applied to this task with mixed success; chromatographic separation of the peptides was clearly helpful, whereas enrichment by metal affinity chromatography met with surprisingly little success. We conclude that locating sites of phosphorylation remains a significant challenge at this level of sample abundance.

Chemistry Techniques, Analytical↗