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| Presenter: |
| Nelli Taranenko |
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| Institute: |
Lab or Branch |
| MassTech R&D, Burtonsville, MD |
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| Title: |
| Atmospheric pressure matrix-assisted laser
desorption/ionization interfaced with hybrid quadrupole/
time-of-flight mass spectrometer analysis of phosphorylated
peptides |
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| Authors: |
| N.I. Taranenko,V.V. Laiko, B.D. Musselman,
V.M. Doroshenko |
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| Abstract: |
| Atmospheric pressure (AP) MALDI mass spectrometry
(MS) was established as useful bioanalytical tool offering
capabilities of tandem MS analysis. Quadrupole/ time-of-flight
(QTOF) MS is another popular type of mass spectrometry
offering high mass resoluton and accuracy in both MS and
MS/MS modes. An AP/MALDI source (MassTech Inc.) equipped
with nitrogen laser (wavelength 337 nm) and x-y linear
stages for multi-array sample analysis was interfaced
with a Micromass Q-Tof2 hybrid quadrupole/ time-of-flight
mass spectrometer. High repetition Photonics Industries
International Model DS10-351-5 Nd:YLF laser (351 nm) was
used to improve the system sensitivity and throughput.
The Nd:YLF laser output was attenuated to 20 uJ/pulse
and coupled with an optical fiber to deliver the laser
energy onto the AP/MALDI target. The optimization of the
Z-spray temperature and other parameters, the electric
field drugging AP MALDI ions toward the Q-Tof2 inlet hole,
and laser parameters allowed reliably detect BSA in low
femtomole range in both MS and MS/MS modes. To demonstrate
the capabilities of AP MALDI-QTOF-MS for phosphopeptides
analysis, experiments were performed on a number of synthetic
phosphopeptides and bovine beta-Casein. Phosphorylated
peptides from tryptic digested beta-Casein were selectively
preconcentrated by Immobilized Ga(III) Metal Affinity
Chromatography (IMAC) by following a protocol, which was
modified for better compatibility with MALDI process.
The IMAC-enriched phosphopeptides fragments were subsequently
analyzed by AP-MALDI operated either in positive or negative
mode. Consistent with the previous vacuum-MALDI reports,
the negative mode of AP-MALDI shows significantly increased
signal intensities for phosphorylated peptide fragments.
All 12 expected phosphorylated sites in beta-Casein was
successfully identified in the negative detection mode,
although most of these fragments did not appear in the
positive ion spectra. The use of the Ga(III) IMAC enrichment
further simplifies identification of the phosphorylated
sites by increasing the relative signal intensities of
the phosphorylated fragments as compared to the corresponded
peptide fragments from a non-enriched protein digest.
Our study shows that acquiring positive and negative ion
spectra by AP-MALDI combined with Ga(III) IMAC enrichment
provides a powerful solution for mapping and positive
identification of phospsphorylation sites in proteins. |
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