![Figure 7. Overview of strategies used for the analysis of phosphorylated proteins. This overview presents only a few of the strategies currently implemented for the analysis of phosphorylated proteins. Readers are referred to reviews that cover in greater detail the variety of technologies used for the phosphoproteome analysis.84,98 The methods used prior to MS analysis are mainly concerned with the enrichment of phosphorylated proteins or peptides. (A) Immobilized metal affinity chromatography (IMAC) relies on the affinity for the phosphate group of metal ions (Fe3+ and Ga3+) that are bound to a chelating resin. Several limitations precluded the straightforward use of this method, such as binding of acidic unphosphorylated peptides that necessitate the esterification of the carboxylic acid groups and difficult elution of peptides with multiple phosphorylation sites. Another affinity purification method routinely used for the detection of sites of phosphorylation involves the use of antibodies against phosphorylated tyrosine residues (C). These have been found more efficient for the enrichment of phosphoproteins than phosphopeptides. Equivalent approaches for the enrichment of proteins or peptides containing phosphorylated serine or threonine are not yet in common use. MS-based techniques are mainly concerned with the selection, isolation, detection, and recognition of the site of modification. Precursor ion scanning has been typically used in a tandem quadrupole instrument. The first quadrupole is set to scan over the appropriate peptide m/z range, collision-induced dissociation (CID) is induced in the second quadrupole (or equivalent), and the third quadrupole is set to selectively transmit m/z 79, corresponding to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{PO}_{3}^{-}\) \end{document}. Therefore, only the peptides that yield the characteristic \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{PO}_{3}^{-}\) \end{document} product ion are detected. A conceptually similar approach may be taken to the detection of phosphotyrosine-containing peptides based on detection of the phosphotyrosine immonium ion.85 Another MS/MS method useful in this context is neutral loss scanning, this time applied in positive ion mode. The first quadrupole is set to scan over the full m/z range, CID is achieved in the second quadrupole, whereas the third quadrupole is set to scan with an offset on the m/z scale of –98/z (where z represents the charge state) relative to the scan of the first quadrupole. Therefore, peptides that lose the neutral H3PO4 (mass 98) are detected. These screening steps may be followed by MS/MS with scanning of all product ions derived from the putative intact phosphopeptide ion.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/103/10/10.1182_blood-2003-09-3295/6/zh80100460910007.jpeg?Expires=1743236857&Signature=r4A~brdZKqhjf0m-jYmAPccXFyfDWlvb9XqXTJqxdImXCDFJietWArhvZ9xM5kxHGoh5yfD8S-7DuU9~nLb0xWhh2sa38yFV5LO6s9B7bFIPe1ll4OyaSfqWqNbNZ05uQZk3J65A370~37IBy3caZqI70TFefjhzIUpZ2XxDOVFEU8YSCKIFzgP09uj6Wcl3vwWMcx3btX7rDSk1R~RukKnJwTbLX-Gb~E2LTD4TU9x9Ty7Yl8uhKka9tLwbMUwc952vMs4DdUqSUFfhV2d6G4ofa0pV7mxqXkyHMwUjOvqK-3n1UZPz4VMn86Lfvu3VyqFp3W42nL8aj4peox98wg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Overview of strategies used for the analysis of phosphorylated proteins. This overview presents only a few of the strategies currently implemented for the analysis of phosphorylated proteins. Readers are referred to reviews that cover in greater detail the variety of technologies used for the phosphoproteome analysis.84,98 The methods used prior to MS analysis are mainly concerned with the enrichment of phosphorylated proteins or peptides. (A) Immobilized metal affinity chromatography (IMAC) relies on the affinity for the phosphate group of metal ions (Fe3+ and Ga3+) that are bound to a chelating resin. Several limitations precluded the straightforward use of this method, such as binding of acidic unphosphorylated peptides that necessitate the esterification of the carboxylic acid groups and difficult elution of peptides with multiple phosphorylation sites. Another affinity purification method routinely used for the detection of sites of phosphorylation involves the use of antibodies against phosphorylated tyrosine residues (C). These have been found more efficient for the enrichment of phosphoproteins than phosphopeptides. Equivalent approaches for the enrichment of proteins or peptides containing phosphorylated serine or threonine are not yet in common use. MS-based techniques are mainly concerned with the selection, isolation, detection, and recognition of the site of modification. Precursor ion scanning has been typically used in a tandem quadrupole instrument. The first quadrupole is set to scan over the appropriate peptide m/z range, collision-induced dissociation (CID) is induced in the second quadrupole (or equivalent), and the third quadrupole is set to selectively transmit m/z 79, corresponding to
