Result-driven strategies for protein identification and quantitation--a way to optimize experimental design and derive reliable results.
Uni- or multidimensional microcapillary liquid chromatography (microLC) matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) approaches have gained significant attention for quantifying and identifying proteins in complex biological samples. The off-line coupling of microLC with MS quantitation and MS/MS identification methods makes new result-dependent workflows possible. A relational database is used to store the results from multiple high performance liquid chromatography runs, including information about MALDI plate positions, and both peptide and protein quantitations, and identifications. Unlike electrospray methodology, where all the decisions about which peptide to fragment, must be made during peptide fractionations, in the MALDI experiments the samples are effectively "frozen in time". Therefore, additional MS and MS/MS spectra can be acquired, to promote more accurate quantitation or additional identifications until reliable results are derived that meet experimental design criteria. In the case of what can be designated the expression-dependent workflow, quantitation can be detached from identification and only peak pairs with biological relevant expression changes can be selected for further MS/MS analyses. Alternatively, additional MS/MS data can be acquired to confirm tentative peptide mass fingerprint hits in what is designated a search result-dependent workflow. In the MS data-dependent workflow, the goal is to collect as many meaningful spectra as possible by judiciously adjusting the acquisition parameters based on characteristics of the parent masses. This level of sophistication requires the development of innovative algorithms for these three result-dependent workflows that make MS and MS/MS analysis more efficient and also add confidence to experimental results.
Graber
Armin
A
Discovery Proteomics and Small Molecule Research Center, Applied Biosystems, Framingham, MA 01701, USA. graberan@appliedbiosystems.com
Juhasz
Peter S
PS
Khainovski
Nikita
N
Parker
Kenneth C
KC
Patterson
Dale H
DH
Martin
Stephen A
SA
D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D010449:Peptide Mapping; D012985:Software Design; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D015003:Yeasts
D005656:Fungal Proteins; D010446:Peptide Fragments