ProteoExtract® Phosphopeptide Enrichment SCIMAC Kit and TiO2 Kit

In eukaryotic cells, post-translational modifications of proteins, such as phosphorylation and dephosphorylation, are involved in numerous metabolic pathways and in the transmission of signals that control proliferation, differentiation, and apoptosis. The disregulation of the tightly controlled balance between phosphorylation and dephosphorylation may lead to serious pathological conditions. Determining the site of phosphorylation is, therefore, important to understanding essential signaling pathways and to gain insight into the molecular basis of diseases.

The identification of phosphorylation sites is routinely accomplished by mass spectrometry (MS). Due to the high complexity of cellular proteome fractions there is a general need for specific and efficient enrichment strategies of phosphorylated peptides before MS. Efficient enrichment strategies compensate for the low stoichiometry of phosphopeptides relative to their unphosphorylated counterparts and for poor ionization and ion suppressioneffects inherent to MS analysis.

Of the different chemical- and affnity-based methods for phosphopeptide capture, no one method is suffcient for enrichment of the entire phosphoproteome. The two different ProteoExtract® Phosphopeptide Enrichment Kits introduced here enrich for different, partially overlapping segments of the phospoproteome, which is especially useful when working with complex samples. As a result of different binding mechanisms, maximum subsets of phosphopeptides are captured, independent of peptide properties like amino acid sequence, additional posttranslational modifcations, and conformational features.

Selective and sensitive enrichment of phosphopeptides from complex mixtures using either the SCIMAC or TiO2 ProteoExtract Phosphopeptide Enrichment Kits.

 

50 pM Angiotensin ~6 fmol/125 mL ~7 pg/125 mL
67 pM Calcineurin substrate ~8 fmol/125 mL ~18 pg/125 mL
140 pM a-Casein ~18 fmol/125 mL ~425 pg/125 mL

 

Figure 1. Selective and sensitive enrichment of phosphopeptides from complex mixtures using either the SCIMAC or TiO2 ProteoExtract® Phosphopeptide Enrichment Kits. A complex peptide mixture derived from a tryptic digest of porcine liver extract was spiked with a-casein and two synthetic phosphopeptides (concentrations given above) and subsequently processed using the optimized materials and protocols from the SCIMAC or TiO2 Kits. Mass spectrometry analysis was performed using an ESI-LC/MS instrument operated in positive mode A. Unprocessed sample, B. Enrichment of phosphopeptides using the SCIMAC Kit, C. Enrichment of phosphopeptides using the TiO2 Kit. Arrows indicate the predominant phosphopeptide ions.

SCIMAC Kit

The SCIMAC Kit uses two sequential batch chromatography steps. First, samples are applied to a strong cation-exchange (SCX) resin. Non-acidic peptides bind to the SCX resin, allowing highly acidic peptides that interfere with downstream purification and analysis to be discarded with the supernatant. The eluted sample from the cation exchange step is applied to the MagPrep® Phosphobind Resin, a unique Zr2+-charged IMAC resin, which selectively captures phosphorylated peptides. The efficiency and specificity of phosphopeptide capture by the SCIMAC Kit is shown in Figure 1B.

TiO2 Kit

The ProteoExtract® Phosphopeptide Enrichment TiO2 Kit uses a novel titanium dioxide material to enrich for phosphorylated species from complex protein mixtures. Titanium dioxide is highly selective for phosphorylated peptides in the presence of abundant non-phosphorylated peptides. The protocol and buffers are optimized to produce high yields of the phosphopeptides. Enrichment and selectivity for phosphopeptides is further improved by using a 2,5-DHB “displacer” concentration that is directly compatible with LC-MS and MALDI-MS analysis. The efficiency and specificity of phosphopeptide capturing by the TiO2 Kit is shown in Figure 1C. After enrichment, phosphopeptide ions (marked with arrows) provided the predominant signals and the majority of non-phosphorylated peptides were removed (low background).

 

Product Features  
Kit components All reagents included: binding, wash, and elution buffers, resins
Kit size 100 samples
Sample type Proteolytic digests of protein samples
Sample amount 2.5 nanomole phosphopeptides; scalable
Processing time 0.5–1 h from sample binding to elution
Downstream applications LC/ESI-MS or MALDI-MS

Materials