Proteins & Mass Spectrometry
Mass spectrometry and
tandem MS/MS
Matrix-Assisted Laser
Desorption Ionization
Time-of-Flight Mass Spectrometry
(MALDI TOF
TOF)
Two Applied Biosystems 4800
Proteomics Analyzers (MALDI
TOF-TOFs), a
Waters Synapt HDMS, an MDS-Sciex
4000 QTrap,
and a reflectron, delayed
extraction MALDI-TOF mass
spectrophotometer (Voyager
DE-PRO, Perseptive Biosystems/Applied
Biosystems) are available for
analyses of protein, peptide,
and oligonucleotide
samples.
With 1D or 2D LC separations,
highly
complex mixtures of
peptides can
be analyzed, for example
proteolytic
digests of whole cell lysates,
serum proteins, or subcellular
protein fractions, allowing
identification of 600-2000
proteins from single samples, as
well as more straightforward
identification of
proteins purified by column
chromatography, by metal
affinity
chromatography, by
SDS-PAGE or 2D gel
electrophoresis, or from
PVDF/nitrocellulose membranes,
with a microliter of sample at
micromolar concentrations
sufficient for analysis. These
types of analysis can be used,
for example, to detect and
analyze phosphoproteins and
other post-translational
modifications, for isozyme
analysis, and for identification
of
disulfide bonds.
Additional analyses
can be
performed using
nanospray
ionization on our MDS/Sciex 4000
QTrap, or electrospray
ionization TOF spectrometry
can be coordinated with the
Huck
Institute Mass
Spec facility at
University
Park.
Quantitation
of
differences in protein amount
between different samples can be
done by iTRAQ isotope tagging of
peptides and 2D-LC separations
(up to 8 separate samples with
600-1200 proteins ID'd and
quantitated in a single
experiment); by metabolic in
vivo labeling of cells in
culture using heavy vs. light
amino acids; or by comparison of
spot densities on either
standard or DIGE-labeled 2D
gels, followed by identification
of spots of interest with mass
spectrometry.
Hybrid Triple
Quadrupole Linear Ion Trap
Mass Spectrometry
(4000 QTrap)
An MDS/Sciex
4000 QTrap instrument with
electrospray (ESI), nanospray,
APCI and photoionization
sources, coupled with either an Agilent
1100 HPLC system or a Waters
NanoAcquity UPLC system flowing
through an Advion TriVersa
nanomate ionizaiton source, plus a PAL CTC autosampler, provides sensitive
and versatile platforms for
analysis of a very wide range of
materials, including small
molecules (e.g., drugs, lipids, glycolipids, hormones)
oligonucleotides, carbohydrates
& glycoproteins, peptides, and
proteins. The instrument is
particularly strong for small
molecules identification and
quantitation, and for detection
of post-translational
modifications of proteins or
peptides such as
phosphorylations.
FEES
for PSU researchers (outside
work is welcome, but fees are
somewhat higher):
(There is no
charge for initial consultations
and feasibility discussions, nor
for training on the Voyager
DE-PROMALDI-TOF self-service
instrument.)
Self-service (Voyager DE-PRO
only): $25 per
hour
Analyses performed by facility
staff:
●Edman
Sequencing
of
peptides or proteins on our
Procise 491 sequencer costs
$15 per
cycle, with a $75 minimum
charge
●Peptide
Synthesis
costs
$20 per amino acid
(0.1 mmol half-scale
synthesis) or
$30 per amino acid
(0.2 mmol full-scale
synthesis).
Mass Spec validation
of MW costs an
additional $15.
●simple
clean, desalted
samples
such as purified proteins or
in-gel digestions of single
spots can be analyzed on our
DE-PRO or 4800 MALDI TOF-TOF
by Mass Spec Core personnel
for
$15 per
sample (mass within
0.05%)
or
$25 per
sample (mass within
0.015% using
external/internal calibrants)
●removal
of interfering salts or
detergents if
necessary
(ZipTips) is an
additional
$10
charge per sample
(recommended for all gel
spot digests)
●Trypsin
or other proteolytic
digestions
prepared
by Mass Spec Core personnel:
$25
set-up fee plus
$15 per
sample digested
(includes ZipTip cleanup/desalting
and plate spotting).
●MS
analysis of
digested and desalted simple
sample (e.g., gel spot),
data-dependent MS/MS
analysis, and
protein identification (computer
analysis of spectra obtained
to identify protein):
$35
●Single
dimension C18 nanoflow-LC
separations and MS plus
MS/MS analysis of a single
more complex sample
(e.g., multiple proteins)
are performed for
$100
(necessary for example to
maximize sequence coverage
when searching for
post-translational
modifications, takes the
place of ZipTip cleanup and
MS MS/MS analysis above)
●Other
Staff time
is charged at
$60 per
hour
●more
complex samples and analyses
such as user-provided
tryptic digests for MudPit
or iTRAQ analyses, or
exhaustive
post-translational
modification analyses, are
between $100 and
$1250 per sample
depending on the extent of
additional processing and
staff analysis time needed. For
example,
MudPit and
iTRAQ or ICAT labeled
samples require
two-dimensional
chromatographic separation
followed by MS and MS/MS
analysis and database
searching.
Please discuss your
specific project and
experimental goals with Dr.
Stanley to determine the
analyses and separations
needed and the price
for these more complex
services.
Again,
consultations,
recommendations for reagents and
procedures, and instructions
are all freely available,
please contact Dr. Bruce
Stanley by
email at bstanley@psu.edu,
or by telephone at (717)
531-5329.
Page
maintained by
Bruce A. Stanley
Last modified
September 09, 2009 11:21 AM
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