> Methods for Protein Identification
Two services are provided. They differ in the number of proteins they can be used to identify in a given sample, and in the dynamic range of protein abundance with which they can cope successfully.
Mass Spectrometry of Simple Mixtures
In this method, digests of single
proteins or simple protein mixtures are subjected to mass spectrometry
without further fractionation. This approach is suitable for
identifying single proteins or proteins present in samples of
up to a dozen components, including protein spots from 2-D gels,
protein bands from SDS gels or isoelectric focusing strips,
and proteins that have been purified by a combination of chromatographic
steps. The limitation in complexity is derived from the circumstance
that mass spectrometers consume sample during the analysis.
Usually, they are programmed to acquire MS/MS spectra for precursor
ions that are determined to give the highest signals during
MS analysis. After some number of MS/MS spectra have been acquired,
the sample is exhausted, and no further components can be identified.
Under such conditions, the most abundant components of a mixture
are usually identified preferentially. Thus the number of proteins
amenable to identification is linked to their dynamic range
of abundance. Model 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA) MALDI-TOF/TOF is used for the analysis of simple mixtures.
Mass Spectrometry of Complex Mixtures
More complex peptide
mixtures are fractionated by one- or two-dimensional chromatography
prior to mass spectrometric analysis so that larger numbers
of components may be identified successfully. This approach
is usually necessary for multisubunit complexes not subjected
to electrophoretic fractionation, immunoaffinity eluates not
subjected to electrophoretic fractionation, whole cell lysates,
and physiological fluids. The simplification of the peptide
mixture present in the mass spectrometer’s
source at any one time alleviates the limitations imposed by
the duty cycle of the mass spectrometer. Many hundreds of proteins
may be identified in single experiment in this way. Separation
of peptides may occur on-line with the mass spectrometer so
that fractionation and mass spectrometry are proceeding simultaneously.
Alternatively, fractions may be collected and stored for later
analysis. Two Thermo linear ion trap mass spectrometers (LTQ and LTQ-XL) coupled to nanoAcquity UPLC's are used for analysis of complex mixtures.
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