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MALDI-MS Peptide Disease Biomarker Screening
MALDI-MS PEPTIDE DISEASE
BIOMARKER SCREENING
A recent publication (Petricoin et al,
2002) suggests that naturally occurring peptide disease biomarkers that
bind to C16 supports may be identified via comparative analysis of
MALDI-MS spectra acquired from comparatively large numbers of disease
versus control serum samples. Since preliminary data obtained in the
Keck Laboratory confirms this approach has merit, a new service is
being offered to bring this technology within reach of our users. This
new Keck Laboratory service includes robotic desalting of the serum and
other biological samples followed by automated MALDI-MS data
acquisition - with the resulting data being made available initially as
a hard copy. Serum or plasma samples (10 microliters each) should be
submitted in a Marsh Bioproducts AB0800 PCR plate and should be ordered
by columns. Specifically, samples should fill the wells A1, B1, C1,
etc., before proceeding to A2. We suggest that experimental and control
samples be placed in alternating order and that at least some samples
be run in triplicate (widely dispersed amongst the sample wells) so
that experimental variability may be evaluated. A minimum of 8 total
samples (experimental and control) per plate is required for this
service which begins by the robotic addition of 5 microliters 0.1% TFA
to each sample. After repeatedly (8x) pulling each sample up into a C18
ZipTip and expelling it back into the original sample well, the C18
ZipTip is washed 5x with 20 microliters 0.1% TFA. Bound peptides are
then eluted from the C18 ZipTip with 10 microliters of 50% acetonitrile,
0.1% formic acid into a new 96 well plate. 2 microliters of the eluent
are then removed, mixed with 0.5 microliters of
alpha-cyano-4-hydroxycinnamic acid matrix in 50% acetonitrile, 0.05%
TFA containing an internal standard of 25 fmoles of bradykinin (M+H C12
monoisotopic mass is 1060.569), and subjected to automated MALDI-MS on
a Micromass M@LDI-R mass spectrometer. The internal bradyknin standard
is then used as a "lock mass" to calibrate the spectrum.
The MALDI-R mass spectrometer automatically
acquires data in positive ion detection. The current mass range being acquired
is 800-3,500 Da. Although the latter mass range is adjustable, it is difficult
to acquire meaningful data below about 800 Da due to interference from the
matrix and, especially on the current reflectron (only) equipped instrument, the
ionization response drops off substantially as the mass range is increased above
about 2,500. Currently, the MALDI-R sums 10 individual laser shots into one
spectra with the laser operating at 20 Hz (i.e., 20 shots/second). Thus, a new
spectra is acquired every ½ second. The laser moves in a random walk around the
target well, acquiring data from a maximum of 20 different locations within each
2 mm diameter well. A spectra is considered "acceptable" if it has a signal of
greater than 2% above background noise, less than 95% of saturation, and if
there is at least one m/z detected between 1,125 Da and 3,500 Da. The MALDI-R is
programmed to accept a maximum of 40 acceptable spectra, but if it sequentially
acquires 4 unacceptable spectra, it will move on to another location within the
same target well. The instrument uses an incrementally increasing laser
percentage to heat up the target spot to acquire acceptable spectra, while still
having the lowest possible laser energy, which will provide the best possible
mass resolution. If the MALDI acquires 20 acceptable spectra at one position, it
will then move to another position in the same sample well, and will acquire
another 20 acceptable spectra, unless interrupted by 4 unacceptable spectra.
Once the MALDI-R has shot (not acquired) 40 acceptable spectra, it will move to
the next sample well. This means there can be a maximum of 40 acceptable spectra
acquired for each sample, and that if at no point it acquires acceptable data,
it will try up to 10 different locations within the same sample target well
before moving on to the next sample. Typically, the resulting hard copy spectrum
will represent the average of 20 to 40 individually acquired spectra.
The expected mass resolution is 14,000 at M+H 2,465 and the mass accuracy is
better than approximately +70 ppm. We recommend that reasonably large numbers
(e.g., >20) of experimental (i.e., disease = case) and control samples from
different patients be analyzed to "average out" biological diversity so that
disease-specific peptide markers may be identified with higher confidence.
Although not yet tested, we believe this service may be equally applicable to
other biological fluids (e.g., amniotic fluid, urine, etc). Please especially
note that samples will be identified only by the plate number (which is assigned
by the Keck Laboratory), the plate name (which is assigned by the user and which
may contain up to 8 letters), and the well identification (e.g, C1). This
service will be further expanded by the Spring of 2003 when our current M@LDI-R
instrument is replaced by a MALDI-L/R instrument which will enable the use of
both linear and reflectron modes of operation - with the linear mode allowing
the mass range analyzed to be extended to 250,000 (depending on the matrix
used). Upon request, the entire (averaged) MALDI-MS spectrum can be provided as
a text file listing of approximately 91,400 m/z versus intensity data points
that is suitable for further analysis - including importing into software
programs like Excel. Algorithms that may be suitable for identifying peptide
disease markers and other analysis and visualization tools are now being
designed and written by the Keck Biostatistics Resource, which is Directed by
Dr. Hongyu Zhao, and all users of this service will have access to these
programs as they become available.
Biomarker
Submission Workbook (36 KB Excel Workbook)
This workbook must be submitted as described on the form before plates of biomarker
samples are submitted along with one of the forms below.
Sample submission forms for the
Peptide Disease Marker Screening Service:
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