This document describes the method of rapid protein characterization using microflow LC/MS/MS techniques. The method involves digestion of a protein followed by microflow LC separation of the peptide fragments. Each peptide fragment is automatically selected for collision induced dissociation (CID) using mass spectrometric techniques, including automated data-dependent scan functions. Protein identification is achieved by correlating uninterpreted protein and peptide tandem mass spectral data with sequences in protein or nucleotide databases using computer algorithms. This method offers the potential for automated high-throughput data analysis and provides a high degree of specificity to database searches due to the unique fingerprint-type pattern displayed by the tandem mass spectrum representing every amino acid sequence.
The document details a technique using automated tandem mass spectrometry (MS/MS) coupled to online microcapillary liquid chromatography for protein identification. This method involves digesting proteins and separating peptide fragments via microflow LC, followed by collision-induced dissociation (CID) of selected parent ions.
Experiments were conducted using an Agilent 1100 Series LC/MSD Trap mass spectrometer coupled to an Agilent 1100 Series Capillary LC system. The study evaluated the technique on a complex mixture of five tryptic digests, demonstrating the ability to identify proteins rapidly without the need for two-dimensional gel electrophoresis.
The digests were combined at a concentration of 1 pmol/pL each.
Separation was performed on an 180 µm ID capillary column at a flow rate of 2 pL/min.
The algorithm correctly identified three of the five proteins present in the complex tryptic digest mixture.