Journal of Experimental Botany, Vol 49, 903-911, Copyright © 1998 by Oxford University Press
E Marentes and M Grusak
The low-molecular-weight (LMW), low-abundance protein composition of lupin
and pea phloem exudates was determined using matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)>
Phloem sap was collected from lupin inflorescence stalks and pods (using
shallow incisions) or pea seedlings (by placing cut stems in an EDTA
solution). Western blot analysis of phloem exudate proteins with either a
polyclonal antibody raised against Ricinus communis
sieve-tube exudate proteins or pea Rubisco antibody revealed that the
collected exudates contained phloem sap, and that contamination with other
plant fluids was negligible. Three matrix combinations were tested to
assess their ability to facilitate protein ionization. Sinapinic acid in
combination with trifluoroacetic acid yielded the cleanest mass spectra,
and revealed an array of LMW proteins ranging from 2 to 10 kDa. For pea
phloem exudate, the addition of protease inhibitors to the exudate
collection solution prevented proteolysis of endogenous proteins; the
inhibitors did not interfere with the detection of proteins. The
sensitivity of this technique was sufficient to detect changes in LMW
phloem peptides throughout plant development in lupin, or to detect
differences in the phloem peptide composition of two genotypes of pea.
Because only limited sample preparation is required, MALDI-TOF-MS is a
useful technique for characterizing complex fluids such as phloem
sap.Keywords: MALDI, mass spectrometry, phloem
protein, Lupinus albus L., Pisum
sativum L.
ARTICLES
Mass determination of low-molecular-weight proteins in phloem sap using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
United States Department of Agriculture/Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030-2600, USA; Corresponding author; e-mail: mgrusak@bcm.tmc.edu
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