Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

 

Introduction

Mass spectrometry (MS) is a powerful technology for study of PTMs, including protein phosphorylation. Due to the low abundance of many phosphoproteins and the relatively poor ionization efficiency of phosphopeptides, specific enrichment of phosphopeptides prior to MS analysis is necessary. At present, numerous phosphopeptide enrichment approaches have been established and applied to complex biological samples. We and others have reported that multi-step phosphopeptide purification methods enable better recovery of phosphopeptide and achieve higher selectivity and sensitivity than stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation.

Method

Plant plasma membranes were isolated from Arabidopsis thaliana (Col-0) leaves using a two-phase partitioning system. The concentration of plasma membrane protein was determined by Bradford assay. Protein was digested with Lys-C for 4 hours and then by trypsin overnight.

The peptide mixture was purified with IMAC, TiO2, CPP, SIMAC (IMAC+TiO2), the combination of CPP and IMAC, and the combination of CPP and TiO2, respectively.

Nano-LC-MS was performed using LTQ-Orbitrap XL and LTQ-Orbitrap-XL/ETD mass spectrometer (Thermo Electron, Bremen, Germany) connected to an EASY nano-LC system (Proxeon Biosystems, Odense, Denmark). In CID mode, multi-stage activation (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany).

Results

We first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A-score algorithm. Among 890 unique peptides, 389 of them were identified as phosphopeptides from IMAC enrichment. From TiO2 enrichment, 131 of 240 identified peptides were phosphopeptides. Since the results are not so satisfactory, we further investigated these samples using the combination of CPP and TiO2 enrichment methods. 1024 phosphopeptides were identified from the combined method, with a efficiency of 90% in this combined method. The results produced from the 3 enrichment experiments were carefully analyzed, and we conclude that the combined method gives better phosphopeptide recovery and higher selectivity. The overlap between the 3 enrichment experiments was quite small. We are currently investigating further combination of enrichment methods: SIMAC enrichment and the combination of CPP and IMAC enrichment. Samples will be analyzed by LTQ-Orbitrap-ETD MS, and the behavior of phosphopeptides on CID mode and ETD mode will be compared.

 

Innovative aspects

Combination of different phosphopeptide enrichment methods