In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca(2+) signalling proteins as putative interaction partners of pollen plasma membrane H(+) ATPases.

Abstract

Membrane proteomics is still very challenging due to the low abundance and poor solubility of membrane proteins. Furthermore, membrane protein interaction studies in a non-model organism like Lilium longiflorum require an unbiased preparation and detection approach. The presented strategy to identify putative interaction partners of the PM H(+) ATPase by using a combination of different biochemical techniques, i.e. in vivo crosslinking, immunoaffinity purification and mass spectrometry without the need of genetic engineering, transformation or other molecular biology techniques can be easily transferred to other protein interaction studies. The well characterised interaction of the PM H(+) ATPase with regulating 14-3-3 proteins served as an intrinsic control to proof the suitability and reliability of the presented strategy, whilst newly identified interaction partners may indicate novel regulation mechanisms of the PM H(+) ATPase.