Peptide Library by Mass Spectrometry

1
Peptide Library by Mass Spectrometry
C. Di Poto1, L. K. Pannell2, R. Jamieson1, J. N.
Baraniuk1 1Georgetown University, Washington,
DC 2Mitchell Cancer Institute, University of
South Alabama, Mobile, AL.

• INTRODUCTION
• Many proteomics studies count the number of
  intact proteins in 2D PAGE. Systems based on
  other separation methods like LC-MS/MS have been
  introduced, but the primary outcome continues to
  be the intact proteins that can be identified.
• Complications with these system arise when
• multiple versions of the same polypeptide have
  been entered with different names and database
  accession numbers
• a peptide sequence is shared by more than one
  protein from a protein family
• there are multiple isoforms of a protein sequence
  in the population and database
• proteins like Immunoglobulin variable regions,
  are assessed
• As an alternative, we are developing a
  Peptide-based system using Microsoft Excel and
  Access.

In general, peptide ions with Mowse Scores
greater than 35 can significantly identify
proteins. However many peptides have lower scores
because of low MS/MS peptide signal intensities
Peptides with scores 35 have been linked to Gene
ID and protein identifications for 241 proteins.
Albumin contributed 1190 peptides. Peptides with
scores between 35 and 25 permitted the
identification of 152 proteins after
verifications of correct peptide protein
matching using PIR. The usually discarded
peptides with scores between 10 and 25 yielded
236 proteins. Many of these had the same
sequences as higher scoring peptides, and so
reinforced the presence of these proteins in our
diverse group of samples. The number of
potential peptides increased exponentially at
Scores 10, but many were verified as false
positive sequences (not in PIR database). Some of
these peptides identified 115 Immunoglobulin
variable and constant regions.
METHODS We incorporated the following mass
spectrometry and MASCOT-analyzed peptide sequence
data (i) human plasma samples with additional
assessments for diabetes-related
posttranslational modifications (PTMs), (ii)
human cerebrospinal fluid analyzed for oxidation
PTMs, and, (iii) media supernatants containing
the secretome from cultured human olfactory
epithelial cells.
WORK IN PROGRESS Peptide sequence data will be
imported into a new Microsoft Access Database,
sorted, and matched to previously identified
peptides. The proteins corresponding to these
peptides will then be collated, and made
available for further analysis. The new
information will be integrated into the peptide
database to increase the total number of peptides
and proteins that can be matched by this
peptide-based search and retrieval archive.
This database incorporates 60,000 plasma 57,765
CSF and 2,362 culture media peptides. Sequences
and protein identities were verified using
UniProt consortium tools.
SUPPORT P50 DC 006760
NIEHS RO1 ES015382 DoD
W81XWH-07-1-0618