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Related Experiment Videos

Identifying proteins using matrix-assisted laser desorption/ionization in-source fragmentation data combined with

D C Reiber1, T A Grover, R S Brown

  • 1Department of Chemistry and Biochemistry Utah State University, Logan 84322-0300, USA.

Analytical Chemistry
|March 10, 1998
PubMed
Summary

Matrix-assisted laser desorption/ionization (MALDI) with in-source decay (ISD) provides peptide sequence data. This method aids in identifying proteins and new genes by searching sequence databases with partial sequence information.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Metastable ion decay in matrix-assisted laser desorption/ionization (MALDI) is a routine method for peptide structure determination.
  • In-source decay (ISD) via delayed ion extraction time-of-flight mass spectrometry (TOF-MS) yields primary sequence data from peptide fragmentation within the MALDI ion source.

Purpose of the Study:

  • To present two approaches for integrating ISD MALDI-generated partial sequence information with protein database searching.
  • To demonstrate the utility of ISD MALDI for identifying proteins and potentially discovering new genes.

Main Methods:

  • Utilizing cyanogen bromide cleavage followed by High-Performance Liquid Chromatography (HPLC) isolation of peptide fragments for ISD MALDI analysis.
  • Performing ISD MALDI fragmentation directly on intact proteins (reduced and non-reduced) to obtain partial sequence data.

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Main Results:

  • ISD MALDI analysis of a 6034.84 Da human apotransferrin fragment yielded two noncontiguous sequence segments (55% of total sequence), enabling unique protein identification in databases.
  • Searching expressed sequence tag (EST) databases with ISD MALDI data demonstrated potential for rapid new gene identification.
  • Fragmentation of intact proteins (human apotransferrin, bovine serum albumin) using ISD MALDI generated 5-7% sequence information, typically from the C-terminus.

Conclusions:

  • ISD MALDI is a valuable tool for generating partial protein sequence information, facilitating protein identification and gene discovery.
  • Combining chemical cleavage with ISD MALDI enhances the amount of sequence data obtainable from larger proteins.
  • Direct ISD MALDI fragmentation of intact proteins offers an alternative strategy for rapid sequence information acquisition, particularly for C-terminal regions.