Automated Analytical System for the Examination of Protein Primary Structure

Abstract

This paper describes an automated analytical system for the examination of protein primary structure in which (i) the target protein is first purified by immunoaffinity chromatography, (ii) subsequent chromatographic and chemical reaction steps in the sequencing process are directly coupled, (iii) buffer exchange between these unit operations is achieved while the protein is absorbed on a mixed bed of strong ion exchange sorbents, (iv) proteolysis occurs in an immobilized trypsin column having a 10-1000 fold-excess of enzyme, (v) the tryptic digest is directly transferred to a perfusion dilute capture column where it is concentrated and rapidly desalted, and (vi) peptides eluted from the dilute capture column and analytical microbore and capillary perfusion reversed-phase chromatography columns are analyzed by either single-stage mass spectrometry (MS) or tandem MS/MS. Protein structure variants were easily recognized, and in the case of hemoglobin (Hb) S, the site of variation from Hb A0 was verified.

Mechanical Injury and Cytokines Cause Loss of Cartilage Integrity and Upregulate Proteins Associated with Catabolism, Immunity, Inflammation, and Repair

“…Peptides were eluted on a gradient from 0 -40% buffer B (10 mM phosphate buffer pH 2.8, 400 mM KCl, 25% acetonitrile) over a 40 min period and then from 40 -95% buffer B over a 10 min period, holding at 95% buffer B for 5 min before returning to the starting conditions (total run time was 80 min). One minute fractions (2-adjacent 0.5 min fractions) were collected over the peptide eluting LC/MS/MS-Fractions were injected manually (Rheodyne; manual injector with 0.5-l internal loop) onto a 75-m inner diameter ϫ 160 mm column with 10-m tip (New Objective) packed in-house with Vydac protein/peptide C18 packing material (5-m particle size, 300 Å pore size) as described previously (28). Peptides were loaded at 2% buffer B (buffer A: 1.2% acetic acid in 98.8% water; buffer B: 1.2% acetic acid in 90% acetonitrile; flow rate: 250 nl/min) for 12.5 min before elution on a 147.5-min gradient from 2% to 40% buffer B followed by a wash out for 20 min from 40% to 60% B and returning to 2% B over 15 min and equilibrating at 2% B for 60 min.…”

Immobilized trypsin systems coupled on-line to separation methods: Recent developments and analytical applications

“…Hsieh et al [32] developed a fully automated five-column chromatography system coupled to an ESI-MS-MS for the isolation, digestion, and characterization of human hemoglobin. The system configuration is reported in Figure 2.A.…”

“…The model system chosen for this study was milk and the total analysis time in the tandem column mode of operation was under 2 h. [32 -34, 36, 38], by permission of the Editor (Copyright 1996 American Chemical Society; Copyright 1996, 2003 Elsevier). A) Five-column chromatography system for the isolation and characterization of human hemoglobins [32]. B) Schematic diagram of the system used to automate reduction, alkylation, proteolysis, and reversed-phase chromatography of proteins [33].…”

Identification of the protein-drug adduct formed between aldehyde dehydrogenase andS-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry

“…12±24 This approach affords advantages over conventional solution digestion in that it minimizes sample loss during handling and transfer, reduces the reaction time required, decreases autolysis since the protease is immobilized on stationary support, and, most importantly, improves protease cleavage efficiency and yields relatively reproducible digestion fragments. 13,15,16,18,19,25 However, this tech-nique has so far largely been used to characterize commercially available protein standards. In this report, on-line proteolytic digestion LC/MS was applied to study specific drug-protein interactions, namely MeDTC-SO, the putative active metabolite of disulfiram, and recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH).…”