Purify phosphorylated peptides from complex protein digests for identification in proteomics research.

The Thermo Scientific Phosphopeptide Isolation Kit is designed to simplify the isolation and identification of phosphopeptides from protein digests through the specific interaction of phosphate groups with immobilized gallium. The gallium-chelated IDA-based resin provides less nonspecific peptide binding and greater specificity for both single and multi-phosphorylated peptides than iron-chelated and NTA-based resins. This kit is compatible with classical methods for phosphopeptide analysis (e.g., 32P radiolabeling, Edman degradation and TLC/HPLC), yet it has been optimized for more sensitive and more powerful LC-MS and MALDI-TOF mass spectrometric methods of phosphopeptide analysis.

Highlights

• High binding capacity – each easy-to-use SwellGel Gallium (III) Disc
  is made of 25 µl gallium (III) resin with a binding capacity for phosphopeptides
  of approximately 150 µg
• Reliable – isolates single and multiple phosphorylated peptides; low
  nonspecific binding
• Ideal for MS analysis – simplifies mass spectrometry analysis by
  isolating phosphorylated peptides from complex mixtures; peptides can
  be eluted with MS-compatible buffers
• Fast – total procedure time is just 15 minutes

Beta-Casein was digested with immobilized trypsin for 4 hours at 37°C and 3 µl (approximately 5 µg) of this digest was adjusted to pH 3.0 by adding 50 µl 0.1% acetic acid. The sample was applied to the column and washed with 75 µl of 0.1% acetic acid, followed by 75 µl of 0.1% acetic acid 10% acetonitrile, and a final wash of 75 µl water. Sample was then eluted in three separate fractions using 20 µl of 100 mM ammonium bicarbonate. Elution # 1 (0.5 µl) was mixed with 0.5 µl of MALDI matrix (saturated a-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid) and 0.5 µl ammonium citrate to improve detection of phosphopeptides. This mixture was spotted and analyzed in positive ion, linear, delayed-extraction mode on an Applied Biosystems Voyager DE-PRO MALDI-TOF mass spectrometer (Figures 1 and 2).

Figure 1 MALDI-TOF mass spectrum of tryptic digest of ß-Casein. Peaks at masses 646, 742, 748, 780, 830 and 2,186 represent the expected peptides from a tryptic digest of ß-casein. Peaks for the known phosphopeptides at 2,910 Da and 3,123 Da are not resolved because of the additional negative charge. The peak at mass 2,909 represents a partial digest fragment. See larger image.

Figure 2. MALDI-TOF mass spectrum of tryptic digest of ß-Casein after sample has been processed with the Phosphopeptide Isolation Kit. The 2,062 peak is the single phosphorylated peptide representing the amino acid sequence from residue 48 to 63. The 3,123 peak is the peptide from residue 16 to 40 containing four phosphate groups. None of the non-phosphorylated peptides of ß-casein are detected in significant quantities.  See larger image.

Selective purification of phosphopeptides from complex mixtures prior to mass spectroscopy (MS)-based analysis improves and simplifies data quality and analysis. The new Pierce Phosphopeptide Isolation Kit achieves this by the specific interaction of phosphate groups to immobilized gallium that is compatible with classical methods for phosphopeptide analysis (³²P radiolabeling, Edman degradation and TLC/HPLC). It has been optimized for more sensitive, higher throughput and more powerful LC-MS and MALDI-MS methods of phosphopeptide analysis. Phosphopeptides can be eluted from immobilized gallium by alkaline conditions such as 0.1 N ammonium hydroxide, allowing direct analysis by MS with no additional sample handling.

Each room temperature-stable kit consists of 30 mini-spin columns each containing a SwellGel Gallium Disc for easy handling. No metal chelating or resin equilibration required.

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