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Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage.
The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.
Duke Scholars
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Related Subject Headings
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Spectrometry, Mass, Electrospray Ionization
- Ribosomes
- Ribosomal Proteins
- Proteomics
- Proteome
- Mitochondria
- Chromatography, Liquid
- Cattle
- Animals
Citation
![Journal cover image](https://secure.syndetics.com/index.aspx?isbn=/lc.gif&issn=1044-0305&client=dukeuniv)
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Spectrometry, Mass, Electrospray Ionization
- Ribosomes
- Ribosomal Proteins
- Proteomics
- Proteome
- Mitochondria
- Chromatography, Liquid
- Cattle
- Animals