FR AB - Science Reference

P29-M
Protein/DNA technology DSL version 2000 is a set of shareware
protocols for sample and data management.
J. Medalle, M. Randesi, B. Imai; Rockefeller Univ., 1230 York Ave.,
Box 105, New York, NY 10021
Physical sample and data management are major concerns for a DNA
sequencing core facility. Especially one with a small staff and limited space
are affected the most. Samples can overtake the limited freezer space. Paper
trail can overfill office and lab spaces. Both can cause a quick turnover of
DNA sequencing staff. DNA Sequencing Lab’s (DSL) version 2000 objectives
are to alleviate repetitive computer tasks for DNA Sequencing staff and eliminate
physical paper trail. DSL 2000 is a set of protocols for sample and data
management. Its major components are comprised of online submittal forms
at http://protein13-pc.rockefeller.edu/, scripts from Apple Script, and standardized
forms and macros from Microsoft Excel 98, and Outlook Express 5.
The DSL funnels the online sample submissions into sample statements for
each investigator. This is a file containing sample IDs, names, account numbers,
primer info, template info, etc. DSL also transforms the web submissions
into logs for prep and for slab or capillary sequencing reactions. These logs
direct the staff on how to process incoming samples. The set of protocols
streamlines the sample flow from the investigator to the machine as well as
the data flow from machine back to the investigator. DSL’s protocols are
“hands on” to help the staff create e-mails for investigator notifications and
“a click of a button” to compress and to transfer investigator’s sample statements
or data files into their web account. DSL 2000 is a freeware application
and can be integrated into other small core facilities with a limited budget.
P31-S
The analysis of complex tryptic peptide mixtures by multidimensional
LC-MS/MS on a hybrid quadrupole orthogonal
acceleration time-of flight (Q-TOF) mass spectrometer.
A. Millar1, C. Hughes1, T. Andresson2, T. Hemesath2, J.I. Langridge1; 1Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ,
United Kingdom, 2deCODE genetics Inc., Reykjavik
Advances in both HPLC and mass spectrometry instrumentation have allowed
the analysis of protein complexes which have not been separated on a two
dimensional gel. These experiments involve separation of the complex digest
mixture by microcapillary liquid chromatography connected to an instrument
capable of data directed switching between the MS and MS/MS modes. Protein
identification is then achieved via databank searching of the ESI-MS/MS,
providing qualitative information on the proteins that are present. Hundreds
of MS/MS spectra can be acquired in a fully automated fashion, resulting in
the identification of significant numbers of proteins, including low copy
number proteins, from a single LC-MS/MS experiment1. If, however, a complex protein mixture is to be investigated then a fractionation
step prior to separation of the peptides on the basis of their hydrophobicity
is advantageous. We have, therefore, adopted a 2D LC-MS/MS approach
using a capillary LC system (CapLC) operating at nanoliter per min
flow rates coupled to a Q-Tof 2 mass spectrometer. By placing a strong cation
exchange (SCX) cartridge followed by a C18 trap cartridge it is possible to
pre-fractionate the peptides before separation on an analytical C18 column.
After loading the sample, discreet fractions are sequentially eluted from the
cation exchange cartridge using a salt step gradient; the eluted peptides are
then retained on the trapping C18 cartridge whilst they are desalted. Finally
the peptides are eluted from the C18 pre-column, at 200 nL/min, onto a 75
�M ID � 10 cm Waters Symmetry analytical column for separation and elution
into the mass spectrometer.
This analytical approach will be discussed with examples where this methodology
has been used for the analysis of standard protein mixtures and for the
analysis of cell lysates and sub-cellular fractions.
1. Yates et al., Nature Biotechnology (1999);17, (7), 676–682.
POSTER ABSTRACTS
ABRF 2001 ABSTRACTS
P30-T
A laboratory information management system for a small
DNA sequencing core facility.
M.J. Miller; NCI, NIH, Bldg 37, Rm 3C28, MSC 4255, Bethesda,
MD 20817-4255
The DNA Sequencing MiniCore facility of the NCI’s Division of Basic Sciences
currently services over 300 investigators. The facility is designed to support
small scale, short-term sequencing. In the past year we ran over 25,000 samples
with an average turnover time of less than one day. While this represents
a 60% increase in samples over the year before, it is still a relatively
modest operation.
Keeping all these users and their data organized, as well as minimizing the
amount of paperwork involved is a major concern. Although several Laboratory
Information Management (LIM) System software packages exist, they
are often too expensive or too inflexible for a small operation such as ours.
I describe here a LIM system built using components of the Microsoft Office
software package. Users enter sample information data into an Internet form
and this data is automatically entered into the database after inspection by
the MiniCore staff. Programs built into the database determine which sample
sets can be run together on the same gel, and what parameters (such as
which virtual filter and which dye/primer set settings) should be used with
each sample. Samples are assigned to a particular gel and the “sample sheet”
for that gel is automatically generated by the database. By keeping track of
when samples are submitted, how many samples there are, and how many
basepairs of read the user requests, the database aids the facility’s operators
in determining sample priority. An email message is automatically sent when
samples have been processed and data deposited in the user’s data-destination
directory. The database also keeps track of charges and automatically
sends this data to the NIH’s accounting system. The facility thus runs in a virtually
“paperless” environment. There are no hardcopy forms to fill out. All
information is maintained by the computer system.
P32-M
Identification of in vivo phosphorylation sites in
Drosophila armadillo by tandem mass spectrometry.
C.S. Raska, R.M. Pope, D. Rubenstein; Univ. of North Carolina at
Chapel Hill, 4 Casabelle Ct, Durham, NC 27713
Phosphorylation is one of the most important reversible modifications of
eukaryotic proteins. Often, proteins which are associated with uncontrolled
cell growth, and ultimately cancer show an anomaly in their phosphorylation/dephosphorylation
pathways. In humans, a protein, �-catenin, plays a
central role in the development, organization, and regulation of epithelial tissues.
Aberrant regulation of �-catenin is associated with malignancies. For
example, alterations in �-catenin gene structure have been identified in colorectal
and breast carcinoma, and in a large number of melanoma cell lines.
Specifically, �-catenin signaling function is constitutively active in many
melanomas due to mutations that remove phosphoresidues at the amino terminus
of the protein. Armadillo, a protein found in Drosophila melanogaster,
is 71% identical to �-catenin at the amino acid level. �-catenin from Drosophila
and from human keratinocytes binds to armadillo and �-catenin,
respectively, and this binding has been found to be influenced by phosphorylation.
Thus, we have been using armadillo as a model system to study
�-catenin regulation. To further define the regulatory function of phosphorylation,
we are using mass spectrometry to map post-translationally modified
amino acids in armadillo. Armadillo was isolated by immuno-affinity and
ion exchange chromatography. 1-D SDS-PAGE, and in-gel tryptic digestion
were performed to produce a mass spectral fingerprint on a triple quadrupole
instrument using nanoelectrospray. MS scans confirmed peptides matching
both armadillo and �-catenin from one gel spot. Neutral loss scans
identified potentially phosphorylated peptides. MS/MS generated enough
sequence coverage to specifically identify phosphorylated residues. We note
that phosphoresidues are located within the region where binding sites for
cadherin, dTCF, and dAPC are located. We plan mutation studies to further
elucidate the role of phosphorylation in these systems.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 195