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MS/MS Protein Identification
MS/MS Protein Identification
This protein identification service is
based on either nanospray MS/MS or LC MS/MS analysis on a Micromass Q-Tof
API mass spectrometer. Following in gel trypsin digestion, the sample
is either desalted using a C18 ZipTip for nanospray analysis or
injected onto a 100 micron I.D. Atlantis C18 column (Waters) for LC
MS/MS analysis. For the nanospray analysis, an internal peptide
calibrant (Glu-fibrinogen, m/z = 785.85 doubly charged species) is
added to the sample at a final concentration of ~ 200 fmol/µl. prior to
desalting on a C18 packed ZipTip which is eluted with 50% acetonitrile,
0.1% formic acid. The eluted sample is then subjected to nanospray
analysis on the Q-Tof. During this approximately 45 min analysis it is
generally possible to obtain from 6-15 MS/MS spectra which are then
individually searched against the NCBI nr database using the Mascot
algorithm. Nanospray MS/MS analysis is typically used for samples at
the very fmol level. Proteins other than keratin (a possible in gel
digest contaminant) and trypsin are identified in ~ 80% of all samples.
For LC MS/MS analysis, 5ul of sample is injected onto (currently) a 100
micron I.D. Atlantis C18 column running at 500nl/min. A long gradient
of 95 minutes is used in order to obtain good peptide separation.
Buffer A consists of 98% water, 2% acetonitrile, 0.1% acetic acid, and
0.01% TFA. Buffer B contains 80% acetonitrile, 20% water, 0.09% acetic
acid and 0.01% TFA. Data dependent acquisition is performed so that the
mass spectrometer switches automatically from MS to MS/MS modes. After
analysis, the MS/MS spectra are searched using the Mascot distiller (to
locate and combine MS/MS spectra) and the Mascot database search
algorithm. In the Keck facility, we consider a protein identified by
either nanospray MS/MS analysis or LC MS/MS when more than 2 peptides
are matched to the same protein in the database (based on accession
number), and when the matched peptides derive from the correct
enzymatic cleavage sites. In both instances, the percentage of samples
where one or more proteins is identified increases significantly when
the protein derives from an organism whose genome has been sequenced
and is well characterized. In every instance where an identification
has been made by either approach and where the investigator has asked
that we proceed with conventional HPLC isolation/Edman degradation, the
identification has been confirmed. The minimum recommended amount of
protein digest for the LC MS/MS and nanospray approaches is about 10-20
fmoles. SDS PAGE-separated proteins submitted for in gel trypsin
digestion followed by MS/MS protein identification should be stained as
described with Coomassie Blue
(preferably) or with digestion compatible silver stain. However,
Coomassie Blue staining is preferred over the silver staining. The
staining preference is based on a large 2D gel experiment done in our
laboratory where the same proteins (~100 spots total) were digested and
identified from a Coomassie Blue stained 2D gel and from a digestion
compatible silver stained gel. In each case, the proteins did digest.
The number of peptides recovered from 50% of the silver stained spots
corresponded well with the number of spots recovered from the Coomassie
Blue stained spots. However, in the other silver stained spots, only
10-15% of the peptides observed in the Coomassie blue stained sample
were recovered. So, in approximately half of the silver stained spots,
the peptide recovery was poorer than from the same Coomassie blue
stained spot (More
Information).
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