Biomedical subjects

Asish Chakraborty

Publications and source records attributed to Asish Chakraborty.

3 recordsLinked to original sources

PubMed2002

Simplification of complex tryptic digests for capillary electrophoresis by affinity selection of histidine-containing peptides with immobilised metal ion affinity chromatography.

This paper reports on the selectivity behaviour of tryptic peptides on a Cu(2+)-loaded immobilised metal ion affinity chromatography (IMAC) support. Ovalbumin was chosen as a model protein for investigation of the selection and separation of histidine-containing peptides by IMAC off-line coupled with capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF). Two of five histidine-containing peptides in addition to some non-histidine-containing peptides from a tryptic digest of ovalbumin were captured by IMAC. To separate and purify the selected peptides, the IMAC sample was analysed by capillary zone electrophoresis (CZE). The sample was not separated by capillary zone electrophoresis, therefore, micellar electrokinetic chromatography (MEKC) using 10-75 mM SDS was used. Analysis of IMAC sample by MEKC, using low concentrations of SDS (10 mM) was characterised by MALDI-TOF. When using SDS at 75 mM, the migration times of reversed-phase fractions of the IMAC sample, were used to identify the peaks. One of the two selected histidine-containing peptides with two histidine residues was identified, analysing the sample by CZE or MEKC.

Amino Acid Sequence↗

PubMed2002

Global internal standard technology for comparative proteomics.

The work described in this paper tests the efficacy of a global isotope labeling (global internal standard technology, GIST) strategy for quantification in proteomics. Using GIST, overexpression of beta-galactosidase in Escherichia coli was identified and quantified. The GIST protocol involved tryptic digestion of proteins from control and experimental samples followed by differential isotopic labeling of the resulting tryptic peptides, mixing the differentially labeled control and experimental digests, fractionation of the peptide mixture by reversed-phase chromatography, and isotope ratio analysis by mass spectrometry. N-Acetoxysuccinimide and N-acetoxy-[2H3]succinimide were used to differentially derivatize primary amino groups in peptides from experimental and control samples, respectively. The relative concentration of isotopically labeled peptides was determined by isotope ratio analysis with both matrix-assisted laser desorption ionization mass spectrometry and tandem mass spectrometry (MS-MS). Peptide masses and sequences obtained by MS-MS were used to identify proteins. MS-MS was found to be uniquely suited for isobaric peptide quantification.

Amino Acid Sequence↗

PubMed2002

Comparative proteomics based on stable isotope labeling and affinity selection.

Disease, external stimuli (such as drugs and toxins), and mutations cause changes in the rate of protein synthesis, post-translational modification, inter-compartmental transport, and degradation of proteins in living systems. Recognizing and identifying the small number of proteins involved is complicated by the complexity of biological extracts and the fact that post-translational alterations of proteins can occur at many sites in multiple ways. It is shown here that a variety of new tools and methods based on internal standard technology are now being developed to code globally all peptides in control and experimental samples for quantification. The great advantage of these stable isotope-labeling strategies is that mass spectrometers can rapidly target those proteins that have changed in concentration for further analysis. When coupled to stable isotope quantification, targeting can be further focused through chromatographic selection of peptide classes on the basis of specific structural features. Targeting structural features is particularly useful when they are unique to types of regulation or disease. Differential displays of targeted peptides show that stimulus-specific markers are relatively easy to identify and will probably be diagnostically valuable tools.

Animals↗