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FT-ICR MS Services
FT-ICR MS Services
Nano-ESI-FTICR
Electrospray ionization is the most commonly used form of ionization coupled to
FTMS system. The current nanospray source configured with our FTMS consumes
about 200 – 250 nL of samples per minute with sensitivity in the femtomole
range. Typical data acquisition is in the order of seconds so the actual sample
consumptions is ~10 nL per data set acquired. Figure 1 shows a sample mass
spectrum collected with nESI source. Electrospray ionization produces multiply
charged species (ideal for multiply charged peptides/proteins) hence increasing
the dynamic mass range that can be detected.
(ESI) High-Resolution (<50KDa)
High resolution is a necessary
tool looking at relatively complex mixture of peptides/proteins on single mass
spectrum without any LC separation. FTMS offers the highest possible resolution
among all mass spectrometers with its maximum peak capacity exceeding that of
conventional HPLC separation greater than 200 fold. The spectrum in
Figure 2 is
a zoomed section of Figure 1 showing the ability to ID multiple peptides with
overlapping isotopic distributions.
(ESI) Accurate mass
FTMS is known for its high mass accuracy due to how mass/charge is detected.
Ions with a certain mass to charge ratio (m/z) is inversely proportional to its
ion cyclotron frequency (higher m/z ~ lower frequency). Since we can measure
frequency very accurately, and the superconducting magnet is very homogeneous,
we can indirectly calculate the mass of a given ion very accurately. See
Marshall et. al. for detailed calculation and derivation of the relationship
between m/z and ion cyclotron frequency [1]. Average mass accuracy for
peptides/proteins within the 500 – 1500 m/z range, and with external
calibration, is within 2-5 ppm range (with the lower ppm for internally
calibrated spectra, >1ppm possibility). High accurate mass increases the
confidence level of peptide/protein identification, and is ideal for structural
determination of organic molecules. Figure 3 shows the use of FTMS to
distinguish between the monoisotopic peak of a deuterated compound from the
second isotopic peak of the non-deuterated compound (mass difference ~0.003 Da).
(ESI) IRMPD
MS/MS
InfraRed MultiPhoton Dissociation (IRMPD) is a form of fragmentation that occurs
in the ICR cell, and provides structural and peptide-sequencing information. For
peptides, b and y' ions are generated
after a short (~100-400 ms irradiation of 40-70% power of a 40W CO2
laser) pulse. Figure 4A shows IRMPD fragmentation on Substance P. IRMPD can also
be used to study structural information of organic compounds (lower detection
limit mass of 120 Da) as seen in Figure 4B. Fragment ions of organic compounds
are generated from IRMPD are mapped to determine possible precursor or
degradation products.
(ESI) ECD
MS/MS
Electron Capture Dissociation fragments ions in the ICR cell by emitting a beam
of electrons (typically < 1eV) to the ion cloud. The exact detailed mechanism of
ECD is still being investigated by many research groups. Generally
c and z· ions are generated. The higher energy that
is used for the fragmentation provides high degree of sequence information that
is ideal for de novo sequencing and PTM analysis. The reason ECD is
preferred for PTM analysis is because the labile modified groups are not
dissociated upon fragmentation of the peptide back bone. Like IRMPD, the
fragmentation is performed in the millisecond timescale, and shows possibility
for online LC MS/MS experiments. Figure 5 shows an ECD spectrum of Substance P.
Top
Down/Protein Structure/PTM
With its high mass accuracy and its capability for multiple fragmentation
techniques such as InfraRed MultiPhoton Dissociation (IRMPD), Collision Induced
Dissociation (CID), and Electron Captured Dissociation (ECD), the FTMS
instrument is well suited for "unique" analysis of "unusual" samples;
particularly in the area of PTM proteins. The initial step for PTM analysis
involves comparison of control vs. modified sample of purified peptide/protein
profiles in a single mass spectrum by nESI. Peaks difference, as determined by
accurate mass, corresponding to proposed modification is targeted for
fragmentation experiment. Depending on the PTM of interest, fragmentation by ECD
typically retains the covalently bound labile modification, and site of
modification is elucidated from fragment ion masses. IRMPD fragmentation, is
complimentary to ECD, and shows a loss of neutral mass corresponding to the mass
of the PTM component(s). Cleavages along the peptide backbone, as in ECD
fragmentation, are also observed.
A typical procedure to determine site of phosphorylation with FT-ICR and its
IRMPD and ECD capabilities, and without the use of prior separation/enrichment
methods (though these methods are advantages to increasing S/N of peaks of
interest for subsequent fragmenation, particularly when stoichiomietry is not
sufficiently high, ie. >25%) is described below. Initially, nESI broadband mass
spectrum of tryptic digest of control (non-phosphorylated) protein is compared
with that of the phosphorylated sample. Differences in spectral peak features
are determined for possibility of phosphorylation modification (as indicated by
peptides differing in mass of 79.966 Da (or multiple of 79.996 Da for multiple
phosphorylation sites). Difference, tentatively identified phosphorylated
peptide peak(s) are targeted for fragmentation by IRMPD and ECD. IRMPD and ECD
are complimentary fragmentation techniques for identification of phosphorylation
site. Neutral loss of HPO3- (79.966) by IRMPD indicates that the
peptide is phosphorylated. Separately, ECD of phosphopeptides provides highly
efficient cleavages of amino acid backbone of peptide without cleaving the
labile phosphoryl-bond. Mapping of product ion masses will elucidate the site of
phosphorylation by indicating product ion(s) which retain the phospho-group.
IRMPD and ECD can be also be used simultaneously to provide both neutral loss
for phospho-peptide ID and non cleavages of phospho-bond to site of
phosphorylation ID.
Simultaneous use of IRMPD and ECD
shows promising future technique in phosphoproteomic profiling of intact
proteins (for proteins <45kDa) by using IRMPD to "untangle" protein for high
efficiency fragmentation of ECD to determine site of phosphorylation of intact
protein.
LC-MS
Introduction of lower complexity samples into the FTMS instrument can be
accomplished either by Electrospray Ionization (ESI or nESI) or Matrix Assisted
Laser Desorption Ionization (MALDI), and transition between these two modes can
be done within minutes. Nano-ESI, due to its low sample consumption rate (with
spray needle id between 5-30μm) and high sensitivity, is normally preferred over
conventional ESI when sample is limited (<20μL, at <10μM). Typical spray
conditions for biological mixture (low picomole/μL conc.) is 15μL/hr of sample
that is in 50:49.8:0.2% methanol:water:acetic acid. Efficiency of the ESI
process is hindered by presence of salts/detergents (<0.01%), therefore sample
with high salt concentration require a simple ZipTip procedure prior to
subjecting to FTMS analysis. If the amount of sample (for peptides, ie in gel
tryptic digest of protein spot from 2D SDS-PAGE) is even more limited, MALDI is
used where 1-2μL of sample is mixed with 1μL of [2,5-dihydroxybenzoic acid (DHB)]
matrix and spotted on MALDI plate. For higher complex (ie. tryptic digest of 4
protein mixture) biological samples, nano-flow HPLC can be coupled to nESI for
additional separation.
For extreme complex biological matrices (tryptic digests of >100 proteins, ie.
cell lysate, serum, etc.), MultiDimensional Protein Identification Technology (MuDPIT)
can be implemented [2] Figure 6 shows a zoomed in staggered plot of one of 9
salt plug elutions from an automated MuDPIT experiment for tryptic digestion of
a 4 protein complex mixture. Typically 20μL of 50-100 ug of protein digest
solution (3<pH <5) are loaded onto a strong cation exchange column, and
subsequently eluted with multi-steps salt elution onto a reversed phase column
where peptides are separated and directly eluted into the FTMS via nESI. We are
currently testing various approaches/modifications of MuDPIT protocols to
optimize the FTMS system for comparative analysis of control vs. "disease"
proteome. The MuDPIT data acquisition has been automated, and we are currently
working to automate the analysis for protein identification from the collected
data.
References
[1] Marshall, AG; Hendrickson, CL; and Jackson, GS; (1998) Fourier Transform Ion
Cyclotron Resonance Mass Spectrometry: A Primer. Mass Spectrometry Reviews,
17, pp.1-35.
[2] Washburn, MP; Wolters, D; and Yates III, JR; (2001) Larg-scale analysis of
the yeast proteome by multidimensional protein identrification technology.
Nature Biotechnology, 19, pp242-247.
Figures
Figure 1. Spectrum of nanospray ESI of tryptic digest mixture of alcohol
dehydrogenase, BSA, carbonic anhydrase, and myoglobin. Spectrum was obtained
from pooled C18 ZipTip elution of 4 proteins mixture that have been tryptic
digested.
Figure 2. Zoomed inset of Figure 1. Note the distinction of monoisotopic
peak of peptide AD, AH, and BG. The average resolving power for these peaks is
~110,000, 2MB data points. This is a “typical” high resolution mass spectrum
obtained for the purposed of protein identification based on tryptic masses from
complex mixture. Extremely high resolution (>1,000,000) can also be obtained. We
highest resolution obtained for protein at mass 8500 is 1.6 million.
Figure 3. Accurate mass for distinguishing mass differences ~0.003Da.
Figure 4A. IRMPD of Substance P.
Figure 4B. IRMPD fragmentation of Bryostatin 2. A schematic diagram
outlines either the degradation of bryostatin-2 (top to bottom) or the synthesis
of bryostatin-2 (bottom to top) is shown under the spectrum.
Figure 5. ECD of Substance P.
Figure 6. Sample stack plots of MuDPIT experiment. The plot is a zoomed
in section of one of nine salt plug elution of a 2D LC MS run. Each row
corresponds to a collected mass spectrum during peptide elution from a 75 um
reverse phase C18 column. Note that average peptides elute within 2-4 spectra.
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