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MALDI-TOF/TOF Tandem Mass Spectrometry AnalysisABI 4800 Mass Spec

Introduction

Our centre offers mass spectrometry (MS)-based protein identification and characterization using a ABI4800 MALDI TOF/TOF™ Analyzer, which is the new-generation tandem time-of-flight MS/MS system that gives all the advantages of MALDI in a flexible, easy-to-use, ultra-high-performance mass spectrometer. This instrument provides ten times greater sensitivity to identify and quantitate low-abundance proteins in complex biological samples. Its QuanTIS™ precursor selector allows the detection and identification of peptides at high resolution with no significant loss in sensitivity. It is the only MALDI TOF/TOF mass spectrometer with an on-axis laser irradiation, enabling sensitivity in the attamole range.


MS-based Protein Identification

The proteomics platform of the Centre for Genomic Sciences provides protein identification services incorporating both peptide mass fingerprinting (PMF) and MS/MS peptide fragmentation with the use of the ABI4800 MALDI TOF/TOF™ instrument.

A. Peptide Mass Fingerprint (PMF)

Single species of protein can be identified by Peptide Mass Fingerprinting (PMF) using MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry) analysis. This service involves the protein sample to be digested whereby the protein chain is cut into fragments using different proteases. These protein fragments (peptides) with different molecular weights are accurately measured using MALDI-TOF spectrometry. A list of these peptide masses is produced to give a “fingerprint”, which is unique to each protein. Identification of a particular protein can then be achieved by submission of the PMF data to the protein-searching database. This technique offers high sensitivity, high throughput and low cost for protein identification. The PMF analysis, in most circumstances, can achieve positive identification for a single protein spot/band. Success rates are variable for proteins from different species. For example, proteins derived from Human or mouse, which have completed genomic information, can achieve up to 80-90% confident identification. On the other hand, species with incomplete genomic information will have a lower chance to get conclusive results.

B. MS/MS peptide fragmentation

This technique analyzes protein digests using tandem mass spectrometry (ABI4800 MALDI-TOF/TOF™). Proteins are processed in the same manner as for PMF and the digested protein is then analysed by MALDI-TOF, generating a full spectrum of mass lists. The most abundant peptide ions (precursor ions) are further subjected to MALDI-TOF/TOF analysis to measure the resulting peptide fragments. The fragmentation process can generate a ladder of sequence ions of the selected peptide, which will be used for database searching. The protein is identified by matching the fragmentation patterns of a peptide with the in-silico fragmentations of all the peptides in a database and an algorithm assigned which gives identification scores.

In CGS, we combine both types of analysis for searching against our in-house MASCOT databases.