High-sensitivity peptide mapping by capillary zone electrophoresis and microcolumn liquid chromatography using immobilized trypsin for protein digestion

Abstract

Procedures for the reduced-scale analysis of proteins by peptide mapping have been developed, allowing peptide maps to be obtained from picomole to femtomole quantities of protein. The use of trypsin immobilized on agarose gel and placed in a small reactor column has made it possible to reproducibility digest as little as 50 ng of protein. This represents a decrease in sample size of approximately 3 orders of magnitude from conventional tryptic digestion schemes. Separations of tryptic digests were accomplished by using either microcolumn high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). Separations of 100 ng (4 pmol) of tryptic digest samples of beta-casein were achieved with microcolumn HPLC, while separations of approximately 2 ng (80 fmol) of beta-casein tryptic digest (from a total sample size of 50 ng) were possible with CZE. Peptide maps from phosphorylated and dephosphorylated forms of beta-casein were readily distinguishable using both separation methods, demonstrating an ability to detect a single amino acid modification in a protein. Relative standard deviations of peak retention or migration times were less than 3% for microcolumn HPLC and less than 1% for CZE.

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).…”

Immobilized microfluidic enzymatic reactors

“…A wide variety of beads made from agarose, polystyrene, glass, etc., has been tested. Cobb and Novotny [39] prepared a reactor from thick-walled Pyrex tubing (30 cm61 mm ID) packed with trypsin-immobilized agarose gel, Wang et al [40] packed microfluidic chip with 40-60 mm diameter beads and Seong and Crooks [41] used 15.5 mm diameter polystyrene microbeads in PDMS chip. The use of polystyrene-encapsulated superparamagnetic beads (2.8 mm diameter) has been explored with commercial CE instrumentation for performing enzymatic and inhibition assays, as well as for analysis of biological molecules such as antigens and substrates.…”

Effect of pH and ionic strength of running buffer on peptide behavior in capillary electrophoresis: Theoretical calculation and experimental evaluation

“…9 the experimental and predicted resolution between peaks 5 and 2 against the ionic strength is shown. Resolution was calculated from electropherograms using the equation (4) where tz and t, are the analysis times for peptides 2 and 5, respectively, and wz and w, their respective baseline peak widths. The agreement between both results is good, indicating the same trend although a small quantitative difference is observed.…”