Mass determination of low-molecular-weight proteins in phloem sap using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The low-molecular-weight (LMW), low-abundance protein composition of lupinand pea phloem exudates was determined using matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)>Phloem sap was collected from lupin inflorescence stalks and pods (usingshallow incisions) or pea seedlings (by placing cut stems in an EDTAsolution). Western blot analysis of phloem exudate proteins with either apolyclonal antibody raised against Ricinus communissieve-tube exudate proteins or pea Rubisco antibody revealed that thecollected exudates contained phloem sap, and that contamination with otherplant fluids was negligible. Three matrix combinations were tested toassess their ability to facilitate protein ionization. Sinapinic acid incombination with trifluoroacetic acid yielded the cleanest mass spectra,and revealed an array of LMW proteins ranging from 2 to 10 kDa. For peaphloem exudate, the addition of protease inhibitors to the exudatecollection solution prevented proteolysis of endogenous proteins; theinhibitors did not interfere with the detection of proteins. Thesensitivity of this technique was sufficient to detect changes in LMWphloem peptides throughout plant development in lupin, or to detectdifferences in the phloem peptide composition of two genotypes of pea.Because only limited sample preparation is required, MALDI-TOF-MS is auseful technique for characterizing complex fluids such as phloemsap.