Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.
This method will yield an enrichment of phosphopeptides that can be quantitatively analyzed. A number of parameters can be altered in this protocol, but the most important aspect to remember is to preserve your phosphorylation. Preparation of cells for quantification can be achieved in a number of different ways, for example if you cannot label your cells in vivo, tags such as ICAT[3] or ITRAC [4] can be used post extraction to differentially label two cultures for quantification purposes.
<p class="jove_c…We would like to thank Dr. Rich Rogers for technical assistance with mass spectrometry analyses and helpful discussions, and Drs. Rob Moritz, Jeff Ranish, and Hamid Mirzai for helpful discussions.
This work was funded by the NIH Centers of Excellence.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Isotec™ L-Arginine-13C6,15N4 | Sigma-Aldrich | 608033 | ||
Isotec™ L-Lysine-15N2 | Sigma-Aldrich | 609021 | ||
GIBCO™ Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid | Invitrogen | 70011044 | ||
SigmaFAST Protease Inhibitor Tablets | Sigma-Aldrich | S8820 | ||
Pierce Halt Phosphatase Inhibitor Cocktail | Thermo Scientific | 78420 | ||
Retsch PM100 Ball Mill Grinder | Retsch | 20.540.0001 | ||
Retsch 125 ml Stainless Steel Grinding Jar | Retsch | 01.462.0148 | ||
Ultramicrospin C18 column | The Nest Group | SUM SS10 | ||
Sep-Pak Vac 500 mg C18 | Waters | WAT043395 | ||
TSK-Gel Amide-80 4.6 mm x 25 cm analytical column | TOSOH BioSciences | 13071 | This is the HILIC chromatographic column. | |
Guard column 4.6 mm ID x1 cm | TOSOH BioSciences | 19021 | We recommend this guard column to extend the life of your HILIC column. | |
PHOS-Select™ Iron Affinity Gel | Sigma-Aldrich | P9740 | This is the IMAC resin. Aliquot and store. |