从生物化学原理的角度讲述了怎样将质谱应用于磷酸化蛋白质组学的研究，需要一张一张地看图，识别磷酸化修饰位点。这需要借助于高精度的质谱

磷酸化修饰最常见于 serine, threonine and tyrosine

FIGURE 1. Structures of phosphorylated serine, threonine and tyrosine residues.

The major goals of mass spectrometry-based phosphoproteomic analysis

(i) identify the presence of phosphorylated peptides,
(ii) determine the identity of phosphopeptides by their amino acid sequences,
(iii) localize the specific sites of phosphorylation, and
(iv) quantitatively determine any temporal dependence to the phosphorylation site occupancies, under the specific biological conditions from which the phosphoprotein
was isolated.

A. Ionization of Phosphorylated Peptides

B. MS Analysis of Phosphorylated Peptide Ions

C. Tandem Mass Spectrometry and Gas Phase Ion Chemistry of Phosphorylated Peptides

1. 最常用的磷酸化蛋白质组学碎裂技术是CID、ECD和ETD。图二显示了可用的碎裂模式。

FIGURE 2. Flow chart of demonstrated tandem mass spectrometry strategies for phosphopeptide analysis.

Multiple MS/MS dissociation methods have been used for the analysis of phosphopeptide precursor ions. Commonly applicable phosphopeptide precursor ion charge states (n) are specified for each MS/MS technique. Some techniques, namely CID-, PSD-, and IRMPD-MS/MS, may involve formation of nonsequence product ions that diagnostically indicate the presence of phosphorylated peptide ions (indicated with arrows connecting ‘‘MS/MS’’ methods to ‘‘diagnostic ions’’). Observation of these diagnostic product ions has also been used to trigger precursor ion activation by other MS/MS methods (i.e., ECD) or MS/MS activation of the resultant neutral loss product ion (e.g., MS3; indicated with arrows connecting ‘‘diagnostic ions’’ to ‘‘MS/MS’’ or ‘‘MS3’’ methods).

2. 碎片离子的命名

FIGURE 3. Nomenclature of peptide backbone fragment ions or sequence ions.

3. Formation of 79 m/z (PO3) and MþnH-79� Ions
   
   FIGURE 4. Multidimensional electrospray MS mapping of in vitro phosphorylation of Sic1p, a regulator of the G1/S transition in budding yeast using a triple quadrupole mass spectrometer.

A: HPLC–ESI-MS selected ion monitoring trace for m/z 79 overlaid with the HPLC-UV trace of a tryptic digest of purified phosphorylated Sic1p.

B:Negative ion mode precursor ion ESI-MS scan for m/z 79 of the LC–MS-fractionated material marked with an ‘‘*’’ in the chromatogram shown in panel A. The presence of five phosphopeptides, each marked with a unique symbol,
were identified in this fraction.

C: Positive ion mode CID-MS/MS product ion spectrum of m/z 637.6, the triply charged ion for the 1909.8 Da phosphopeptide marked with a ‘‘黑色三角’’ in panel B. Reproduced from Annan et al. (2001) with permission from American Chemical
Society, copyright 2001.