Olga A. Mirgorodskaya

Quantitation of peptides and proteins by matrix- assisted laser desorption/ionization mass spectrometry using 18 O-labeled internal standards

A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards for the tested sample by enzymatic hydrolysis of the same sample (with known concentration) in (18)O-water. A mathematical algorithm was developed which uses the isotopic patterns of the substance, the internal standard, and the substance/internal standard mixture for accurate quantitation of the substance. A great advantages of the proposed method is the absence of molecular weight limitation for the protein quantitation and the possibility of quantitation without previous fractionation of proteins and peptides. Using this strategy, the peptide angiotensinogen and two proteins, RNase and its protein inhibitor, were quantified by MALDI-time-of-flight (TOF) mass spectrometry.

Primary structure of three cationic peptides from porcine neutrophils: Sequence determination by the combined usage of electrospray ionization mass spectrometry and Edman degradation

The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.

Novel repressor of the human FMR1 gene − identification of p56 human (GCC)n-binding protein as a Krüppel-like transcription factor ZF5

A series of relatively short (GCC)n triplet repeats (n = 3–30) located within regulatory regions of many mammalian genes may be considered as putative cis‐acting transcriptional elements (GCC‐elements). Fragile X‐mental retardation syndrome is caused by an expansion of (GCC)n triplet repeats within the 5′‐untranslated region of the human fragile X‐mental retardation 1 (FMR1) gene. The present study aimed to characterize a novel human (GCC)n‐binding protein and investigate its possible role in the regulation of the FMR1 gene. A novel human (GCC)n‐binding protein, p56, was isolated and identified as a Krüppel‐like transcription factor, ZF5, by MALDI‐TOF analysis. The capacity of ZF5 to specifically interact with (GCC)n triplet repeats was confirmed by the electrophoretic mobility shift assay with purified recombinant ZF5 protein. In cotransfection experiments, ZF5 overexpression repressed activity of the GCC‐element containing mouse ribosomal protein L32 gene promoter. Moreover, RNA interference assay results showed that endogenous ZF5 acts as a repressor of the human FMR1 gene. Thus, these data identify a new class of ZF5 targets, a subset of genes containing GCC‐elements in their regulatory regions, and raise the question of whether transcription factor ZF5 is implicated in the pathogenesis of fragile X syndrome.

Bradykinin degradation pathways in human blood plasma

The kinetics of proteolysis of bradykinin and its metabolites in human blood plasma was studied, the limiting stages were determined and each stage of the degradation was correlated with the appropriate plasma enzyme. A scheme of the pathways of major and minor degradation processes has been proposed.

Absolute Quantitation of Proteins by Acid Hydrolysis Combined with Amino Acid Detection by Mass Spectrometry

Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here, we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation. Quantitation of less than 10 fmol of protein standards with errors below 10% has been demonstrated using this method.

Comparative Study of the Action of Bovine Duodenal Proteinases (Duodenases) on Polypeptide Substrates

A comparative study of substrate specificity of bovine duodenal proteinases—chymotrypsin-like duodenase (ChlD) and dual-specificity duodenase (dsD)—was carried out using oligopeptide substrates (human proinsulin, glucagon, melittin, angiotensinogen fragment 1-14). ChlD displayed mainly chymotrypsin-like properties towards these substrates, hydrolyzing peptide bonds carboxy-terminally to bulky aliphatic or aromatic residues. In melittin, ChlD additionally cleaved peptide bonds after Thr and Ser residues. Dual-specificity duodenase (dsD) significantly restricted its specificity to only trypsin-like or only chymotrypsin-like or displayed full activity, combining both specificities, depending on substrate. Both ChlD and dsD efficiently hydrolyzed a single peptide bond (Phe8–His9) in angiotensinogen fragment 1-14. The kinetic parameters of angiotensinogen fragment 1-14 cleavage by ChlD and dsD were determined (kcat/Km = 80,500 M-1·sec-1 for ChlD and 103,000 M-1·sec-1 for dsD).