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Title: MASS SPECTROMETRIC ANALYSIS OF IAA PROTEIN CONJUGATES IN ARABIDOPSIS AND STRAWBERRY

Author
item PARK, SEIJIN - U. MINNESOTA
item HIGGINS, LEEANN - U. MINNESOTA
item Slovin, Janet
item COHEN, JERRY - U. MINNESOTA

Submitted to: Pacifichem Symposium
Publication Type: Abstract Only
Publication Acceptance Date: 4/22/2005
Publication Date: 12/15/2005
Citation: Park, S., Higgins, L., Slovin, J.P., Cohen, J. 2005. Mass spectrometric analysis of iaa protein conjugates in arabidopsis and strawberry. Pacifichem Symposium.

Interpretive Summary:

Technical Abstract: Indole-3-acetic acid (IAA) is a plant hormone that has many important regulatory roles in development and growth. The conjugation of IAA to different molecules, including peptides and proteins, is a common feature of higher plants. We prepared antibodies against the IAA moiety and detected immunoreactive proteins in crude extracts of Arabidopsis (Col-0) seeds and strawberry (F. vesca) fruit. To verify the presence of conjugated IAA, extracts were hydrolyzed and the amount of free IAA released was analyzed by GC-MS. The immunoreactive proteins were partially purified and their tryptic digests were analyzed using precursor ion scans and tandem MS scans. To interpret those spectra we analyzed IAA-amino acids and synthetic IAA-peptides and found that m/z 130 and 158 were signature ions for IAA-peptides. Our interpretation was further aided by the comparison of precursor ion scans and MS-MS scans. In precursor ion scans, we monitored 2 signature ions. However, we have observed similar ions in some MS-MS spectra of non-IAA containing peptides, including lysine-containing peptides. We could exclude some of those false signature ions by comparing the theoretical and experimental isotope envelopes, specifically the ratios of isotopically-related peaks, at m/z 130. The MS-MS spectra were processed with Pro ID. If Pro ID assigned non-IAA-containing peptides to our protein of interest with high confidence, we excluded the MS-MS from further analysis. Modification to peptides with "any mass up to" 160 Da was considered during the Pro ID searches. If Pro ID found a peptide matched with a modification of 158 Da, and there was a positive precursor ion scan for m/z 130 or 158, or if there was no match to an MS-MS from Pro ID but positive precursor ion scan, we interpreted the MS-MS spectra manually. In this way, we are characterizing two immunoreactive Arabidopsis proteins and a strawberry protein for sequence and site(s) of IAA conjugation.