Absolute and Site-Specific Quantification of Protein Phosphorylation Using Integrated Elemental and Molecular Mass Spectrometry:  Its Potential To Assess Phosphopeptide Enrichment Procedures

An ICP-MS, ESI-MS and molecular modelling investigation of homogeneous gallium affinity tagging (HMAT) of phosphopeptides

“…[58,59] Peptides containing phosphorylated serine and threonine residues can be enriched and modified by methods based on Isotope Coded Affinity Tags, [10,60] in which the phosphate group is eliminated by base hydrolysis and a biotin or mercaptoethanol group is introduced by Michael addition, then selectively detected by capture/elution with streptavidin or with sulphur specific antibodies.…”

“…[1] The characterisation of phosphorylation sites has been widely investigated by the use of tandem mass spectroscopy. [2][3][4][5][6][7][8][9][10][11] A typical phosphoproteome work-flow involves the purification of the protein of interest by gel electrophoresis or reversed phase chromatography, trypticdigestion, enrichment of phosphopeptides followed by analysis using liquid chromatography coupled to tandem mass spectrometry. Phosphopeptide identification by triple quadrupole mass spectrometry is achieved by two methods; neutral loss scans that identify phosphopeptides from the loss of H 3 PO 4 or HPO 3 on collisional activation from phosphorylated serine and threonine residues or parent ion scans for the common product ions H 2 or PO 3 -.…”

ICP‐MS‐Based strategies for protein quantification

“…The measured degree of phosphorylation of b-casein and regular a-casein were in good agreement with the structural data of the proteins. Helium was also used as the cell gas to minimize polyatomic interferences during the phosphorus determination (Pröfrock et al, 2005;Navaza, Encinar, & Sanz-Medel, 2007a;Navaza et al, 2008).…”

“…The capillary HPLC-ICP-MS method showed excellent linearity over three orders of magnitude, and provided an adequate detection limit of P (110 fmol, 3.4 pg P). Accurate quantification of the phosphopeptides in a tryptic digest of acasein and b-casein from bovine milk was implemented (Navaza, Encinar, & Sanz-Medel, 2007a;Navaza et al, 2008).…”

Recent Developments in Mass Spectrometry Analysis of Phosphoproteomes

“…Determining the absolute amount of an analyte in a sample is a highly desirable goal (Pereira Navaza et al, 2008). An LC-ICP-MS protocol has been developed to achieve this goal.…”