This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ECD is known to generate isoAsp-specific diagnostic ions for isoAsp identification and quantitation. Proteome-scale isoAsp mapping has also been demonstrated using a nano liquid chromatography (nanoLC)-ECD method. However, ECD only works with multiply-charged cations that are typically generated by electrospray ionization (ESI). Some peptides are not efficiently ionized by ESI, particularly in the presence of ionization suppression agents. Further, the elution time of a given peptide may be too short to allow sufficient signal averaging for the observation of diagnostic ions. Finally, offline LC-ESI-ECD analysis typically requires picomoles of sample, which are not always available for samples of clinical origin. Matrix-assisted laser desorption/ionization (MALDI) offers a complementary ionization method that can be used to improve the protein coverage and when sample amount is limited. Comparing with ESI, MALDI is less susceptible to ionization suppression. As an offline technique, there is no time constraint for signal averaging for analyses of MALDI-generated ions. Tandem MS analysis with femtomoles of sample is also possible with MALDI. However, MADLI produces predominantly singly-charged ions, which cannot be analyzed by ECD due to its charge-reducing nature. We are currently exploring alternative methods for isoAsp analysis of MALDI-generated ions. One such method is in-source decay (ISD), which produces predominantly c- and z-type ions, presumably initiated by hydrogen radical transfer from the matrix to the analyte ions. Preliminary data shows ISD works well on the Bruker Reflex IV MALDI-TOF instrument. Peaks with mass corresponding to those of diagnostic ions were also observed in the ISD spectrum of a synthetic isoAsp-containing peptide, although they were located at a spectral region (<1000 Da) where interference peaks of matrix clusters abound. We are currently exploring ways to reduce the matrix cluster interference.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR010888-15
Application #
8365576
Study Section
Special Emphasis Panel (ZRG1-BCMB-H (40))
Project Start
2011-06-01
Project End
2012-08-09
Budget Start
2011-06-01
Budget End
2012-08-31
Support Year
15
Fiscal Year
2011
Total Cost
$3,072
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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