Journal ArticleDOI
On-column digestion of proteins in aqueous-organic solvents.
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TLDR
A method that is capable of rapid on-line digestion and lending itself to high-throughput identification of proteins is demonstrated, applied to the digestion of human transferrin, a proteolytically resistant protein.Abstract:
Proteolytic digestion is an important step in protein identification by peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing. Traditional methods of protein digestion require extended incubation times and have difficulty with proteolytically resistant proteins. Here, we describe a method in which a protein solution was combined with a mixed aqueous-organic solution (methanol, isopropanol, or acetonitrile) and passed through a microcolumn containing immobilized trypsin. Myoglobin sequence coverage was high (>85%) in all three solvents, and differences in spectra were seen among the different solution conditions. Notably, methanol-based digestions produced fewer missed cleavages while acetonitrile-based digestions produced the most peptides and the most intense mass spectra. Flow rates through the column were varied from 0.5 to 15 micro L/min, corresponding to column residence times of 78 and 2.6 s, respectively. All flow rates produced high sequence coverage of myoglobin, although, at higher flow rates, more missed cleavages were observed. No significant increase in undigested myoglobin was observed with flow rates up to 15 micro L/min. The described method was applied to the digestion of human transferrin (hTf), a proteolytically resistant protein. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis detected 42 peptides covering 46% of the hTf sequence. The traditional aqueous method resulted in 12 peptides (8% sequence coverage) only when high concentrations of trypsin were used. Lastly, digestion of low nanomolar myoglobin was shown to produce detectable peptides and resulted in a correct database hit. Thus, we demonstrate a method that is capable of rapid on-line digestion, thereby lending itself to high-throughput identification of proteins.read more
Citations
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Journal ArticleDOI
An Automated and Multiplexed Method for High Throughput Peptide Immunoaffinity Enrichment and Multiple Reaction Monitoring Mass Spectrometry-based Quantification of Protein Biomarkers
TL;DR: A method for an automated magnetic bead-based platform capable of high throughput processing is developed and it is demonstrated that enrichment of peptides from larger volumes of plasma can extend the limits of detection to the low pg/ml range of protein concentration.
Journal ArticleDOI
Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems.
TL;DR: This report is the first using mass spectrometry data to show a linkage between digestion solvent and trypsin specificity, resulting in smaller numbers of semitryptic peptides than an overnight digestion protocol using an aqueous solvent.
Journal ArticleDOI
Effects of modified digestion schemes on the identification of proteins from complex mixtures.
TL;DR: Modifications to a standard digestion protocol demonstrate large, reproducible improvements in protein identification, a result consistent with digestion being a limiting factor in the efficiency of protein identification.
Journal ArticleDOI
Ultra fast trypsin digestion of proteins by high intensity focused ultrasound.
TL;DR: Proteolytic digestion of proteins in seconds under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) has been achieved and greatly reduces the time needed for protein digestion, is of easy implementation, environmental friendly, and economic.
Journal ArticleDOI
Highly efficient enzyme reactors containing trypsin and endoproteinase LysC immobilized on porous polymer monolith coupled to MS suitable for analysis of antibodies.
TL;DR: With the use of this system, immunoglobulin G was digested at room temperature in 6 min to an extent similar to that achieved with soluble enzyme at 37 degrees C after 24 h.
References
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Journal ArticleDOI
An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics
TL;DR: An automated method for shotgun proteomics named MudPIT, which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry, improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.
Journal ArticleDOI
Proteomics: the move to mixtures
Junmin Peng,Steven P. Gygi +1 more
TL;DR: Current approaches to identify and characterize large numbers of proteins and measure dynamic changes in protein expression directly from complex protein mixtures (total cell lysates) are addressed.
Journal ArticleDOI
On Protein Denaturation in Aqueous−Organic Mixtures but Not in Pure Organic Solvents
TL;DR: FTIR spectroscopy was used to quantitatively assess the secondary structure of proteins in aqueous−organic mixtures ranging from pure water to a pure solvent and found that the protein secondary structure was much more native-like in pure organic solvents than in most water−solvent mixtures.
Journal ArticleDOI
Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices.
TL;DR: The proteolytic activity of the enzymatic microreactor on chip was demonstrated at different flow rates with the cleavage of fluorescently labeled casein used as a substrate, and the excellent performance of the monolithicmicroreactor was demonstrated with the digestion of myoglobin at the fast flow rate of 0.5 microL/min.