Multidimensional Strategy for Sensitive Phosphoproteomics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO₂ Chromatography Applied to Proximal EGF Signaling

Abstract

Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to sample loss, and thus, phosphoproteomics studies are usually only performed on highly abundant samples. Here, we present a comprehensive phosphoproteomics strategy applied to 400 μg of protein from EGF-stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into monophosphorylated and multiphosphorylated pools using sequential elution from IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and nonphosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4700 unique phosphopeptides, while 636 phosphosites were changing following short-term EGF stimulation, many of which were not previously known to be involved in EGFR signaling. We further compared three different data processing programs and found large differences in their peptide identification rates due to different implementations of recalibration and filtering. Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that, despite having highly accurate precursor mass determination, peptides with low fragment ion scores should not automatically be reported in phosphoproteomics studies.

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

“…phy (21)(22)(23)(24) and immunoprecipitation (36), respectively. Prior to LC-MS/MS analysis, all iTRAQ ® labeled samples were prefractionated offline on a TSKGel Amide 80 HILIC HPLC column.…”

“…Phosphopeptide Enrichment-TiO 2 chromatography was used to enrich samples for phosphopeptides as described previously (21)(22)(23)(24) with minor modifications. Sample (130 g) was resuspended in 80% acetonitrile (ACN) (v/v), 5% (v/v) trifluoroacetic acid (TFA), 1 M glycolic acid in a polypropylene tube containing 1 mg TiO 2 beads and incubated with constant shaking for 30 min.…”

The Global Phosphoproteome of Chlamydomonas reinhardtii Reveals Complex Organellar Phosphorylation in the Flagella and Thylakoid Membrane

“…In contrast to these findings, the relative abundance of pTyr is typically observed to range from 1.5% to 2.4% in a variety of organisms (86 -89). This result likely stems from differences in methodology and organism used; for example, in HeLa cells, observed pTyr abundances range from 0.7-1.8% (86,90).…”

Comprehensive Quantitative Comparison of the Membrane Proteome, Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells

“…Enrichment of Phosphopeptides and Formerly Sialylated Glycopeptides-To separate multi-and monophosphorylated peptides from formerly sialylated glycopeptides and nonmodified peptides, the samples were subjected firstly to SIMAC enrichment (31) to isolate multiphosphorylated peptides, followed by an optimized TiO 2 enrichment procedure (32)(33)(34) to purify monophosphorylated and SA containing glycopeptides from nonmodified peptides. After enzymatic deglycosylation of the TiO 2 bound peptide fraction, HILIC was used to separate the deglycosylated and phosphorylated peptides.…”