Spotlight Laboratory Consumables Why Is Phosphopeptide Enrichment Important?
Ylva Laurin, Ann-Marie Nissfolk, Johan Öhman, Ulrika Meyer, Helena Hedlund and Marianne Albenius GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden,
Ylva.Laurin@
ge.com, + 46 18 6121314
Studying Cancer Cell Signalling Using
TiO2 Mag SepharoseTM Magnetic Beads Phosphorylation is a common reversible post-translational modification involved in the regulation of many essential biological processes, for example cell signalling, which is of prime importance for the study of various disease states such as cancer. The phosphoproteins and phosphopeptides resulting from these processes are important to the understanding of tumour progression, however, there are various barriers to their study. Within the cellular environment, phosphopeptides are usually transient and found in very low concentrations. In addition when proteins are phosphorylated they attain a negative charge and, compared to their non- phosphorylated counterparts, they are poorly ionised which further complicates their measurement using mass spectrometry. Therefore, determination of their relative levels as a function of disease progression is an ongoing challenge for researchers. One approach is to use a simple enrichment technique that ‘concentrates’ the phosphopeptides in samples to aid detection using standard mass spectroscopy approaches. This paper outlines how such a simple enrichment process using TiO2 Mag SepharoseTM magnetic beads was developed, validated using a model sample, and tested in a complex sample derived from a leukaemia cell line.
About the TiO2 Mag Sepharose magnetic beads TiO2 Mag Sepharose magnetic beads use titanium dioxide (TiO2)-based chromatography to simplify the capture and enrichment of phosphopeptides. TiO2 has a high affinity for phosphopeptides and provides efficient enrichment of these from complex samples. When the tubes are placed in MagRack, the magnetic beads are attracted to the magnet within a few seconds. This allows fast removal of the supernatant whilst the beads remain in the tubes. The easy visibility of the beads ensures reliable collection of all the targeted peptides.
Table 1. Phosphopeptide enrichment protocol
1. Equilibration with binding buffer (1 M Glycolic acid, 80% Acetonitrile, 5% Trifluoroacetic acid). 2. Sample application and incubation with mixing for 30 min.
3. Washing with 1 × binding buffer and 2 × wash buffer (80% Acetonitrile, 1% Trifluoroacetic acid).
4. Elution of the target peptides (i.e., phosphopeptides) with elution buffer (5% Ammonium hydroxide).
Study 1: Investigation of the Effective Binding of Phosphopeptides to TiO2 Mag Sepharose Magnetic Beads
To demonstrate the affinity of TiO2 to phosphopeptides, an initial binding study was carried out in which four pure phosphopeptides with masses ranging from 1126.8 to 2192.4 (AnaSpec, Inc), a Phosphate Colorimetric Assay Kit (BioVision, Inc.) and a Shrimp Alkaline Phosphatase (USB) were used to investigate the amounts of bound phosphopeptides to the TiO2 Mag Sepharose magnetic beads. The amounts of bound protein were determined using absorbance at 650 nm (SpectraMax Plus 384, Molecular Devices) and the data given in Table 2. On average, 71% of added phosphopeptides were bound to the beads.
Table 2. Describes amount of phosphopeptides with different MW bound to 10 µl TiO2 magnetic beads.
Figure 1. MALDi-ToF spectra of tart materials with α-casein, β-casein and BSA (A), the corresponding eluate (B) and the eluate diluted 100 times (C). The enriched
phosphopeptides found in B and C are marked with *and also metastable phosphopeptide is seen, marked with a dot.
Study 3: Complex Sample Study to Investigate Enrichment of Phosphorylated
Peptides From a Human Leukaemia Cell Line In a complex sample study carried out by a multidisciplinary group* at Uppsala University, the aim was to map the phosphorylation pattern, of digested proteins from a human leukaemia cell line proteins expressing the BCR-ABL oncogene.
In this study two separate batches of trypsin-digested lysate from human cells were prepared for MS analysis. The first batch used the complete protocol outlined in Figure 2 to prepare an enriched sample, whilst the second batch was processed without the enrichment step as a control sample. The levels of phosphopeptides were then measured and compared. Phosphopeptides were only detected in the material that had undergone enrichment. The MS analysis identified 15 phosphopeptides and 14 phosphorylation sites were found, which are listed in Table 2. A total of 16% of proteins of the leukaemia cell line were phosphorylated in the enriched sample.
Study 2: Model Sample Study: Phosphopeptide Enrichment From
50 pmol -Casein, -Casein, and BSA The aim of this study was to show enrichment of a model sample system using
known quantities of phosphorylated proteins (α-casein and β-casein) in a background of nonphosphorylated bovine serum albumin. A mixture containing 50 pmol of the two forms of casein and 50 pmol non-phosphorylated bovine serum albumin (BSA), was trypsin digested and applied to the TiO2 Mag Sepharose magnetic beads following the same protocol. The eluates with enriched phosphopeptides were then lyophilised, dissolved in 20% Acetonitrile with 0.1% Trifluoroacetic acid and analysed with MALDI-ToF MS (Autoflex III Smartbeam, Bruker Daltonics, Germany). The resultant MS spectra are given in Figure 1, in which the enriched phosphopeptides found in B and C are marked with*, also metastable phosphopeptide is also seen, marked with a dot. Enrichment of
phosphopeptides was achieved with both α-casein and β-casein phosphopeptides found including the tetraphosphopeptide from β-casein. Even when the eluate was diluted a 100 times the two phosphopeptides could still be detected.
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