A solid phase extraction-based platform for rapid phosphoproteomic analysis

Abstract

Protein phosphorylation is among the most common and intensely studied post-translational protein modification. It plays crucial roles in virtually all cellular processes and has been implicated in numerous human diseases, including cancer. Traditional biochemical and genetic methods for identifying and monitoring sites of phosphorylation are laborious and slow and in recent years have largely been replaced by mass spectrometric analysis. Improved methods for phosphopeptide enrichment coupled with faster and more sensitive mass spectrometers have led to an explosion in the size of phosphoproteomic datasets. However, wider application of these methods is limited by equipment costs and the resultant high demand for instrument time as well as by a technology gap between biologists and mass spectrometrists. Here we describe a modified two-step enrichment strategy that employs lysC digestion and step elution from self-packed strong cation exchange (SCX) solid phase extraction (SPE) columns followed by immobilized metal ion affinity chromatography (IMAC) and LC-MS/MS analysis using a hybrid LTQ Orbitrap Velos mass spectrometer. The SCX procedure does not require an HPLC system, demands little expertise, and because multiple samples can be processed in parallel, can provide a large savings of time and labor. We demonstrate this method in conjunction with stable isotope labeling to quantitate peptides harboring >8000 unique phosphorylation sites in yeast in 12h of instrument analysis time and examine the impact of enzyme choice and instrument platform.

Fig. 1. Solid phase extraction-based strong cation exchange chromatography for phosphopeptide analysis

(A) An overview of a typical SILAC SCX/IMAC experiment showing all major sample-handling steps. (B) Hand-packing SPE/SCX cartridges. Empty 6 ml syringe barrel columns fitted with a bottom frit are filled with 1 g polySULFOETHYL A and sealed with a second frit. (C) A step gradient elution protocol for SPE/SCX peptide separation. Including the flow-through, seven discrete 6 ml salt elution steps are used to separate whole-cell extract derived peptides by charge state prior to IMAC phosphopeptide enrichment.

Fig. 2. Solid phase extraction-based strong cation exchange

(A) Shown is the solution charge state distribution of 38,487 phosphorylated and unphosphorylated peptides from lysC digested yeast whole-cell extract subjected to the described SPE/SCX/IMAC enrichment protocol analyzed by LC-MS/MS on a LTQ Orbitrap Velos. (B) The peptide overlap between SCX fractions is shown as the fraction of all peptides that were identified in 1, 2, 3, or more different fractions. (C) A comparison of the solution charge states of the identified phosphopeptides from trypsin and lysC digested yeast whole cell extracts.

Fig. 3. Quantitative phosphoproteomic analysis of a 1:1 mix of “heavy” and “light” labeled wild-type yeast

(A) A summary of identified peptides, phosphopeptides and proteins. Peptide FDR = 0.1%. Protein FDR = 1%. (B) Unphosphorylated (gray) and phosphorylated peptides (blue) per fraction. (C) Unique phosphopeptides per fraction (bars) and a cumulative count of unique phosphopeptides (solid line). (D) The distribution of log₂ peptide abundance ratios (Heavy:Light) for 27,754 phosphopeptides. SD = 0.35.

Fig. 4. The impact of instrument platform and enzyme choice on phosphoproteomic analysis

In both (A) and (B), phosphorylation site counts are shown for each analyzed fraction (bars) along with cumulative counts across fractions (solid lines). (A) A comparison of the same SPE/SCX/IMAC phosphopeptide analysis of lysC digested yeast whole-cell extracts shown in Fig. 3C performed on the LTQ Orbitrap Velos (shown in red) with analysis of the same samples run on a LTQ Orbitrap Discovery (blue). (B) Equal aliquots of yeast extract were digested with lysC (red) or trypsin (blue) and subject to SPE/SCX/IMAC analysis on a LTQ Orbitrap Discovery.