Mapping and analysis of phosphorylation sites: a quick guide for cell biologists

Abstract

A mechanistic understanding of signaling networks requires identification and analysis of phosphorylation sites. Mass spectrometry offers a rapid and highly sensitive approach to mapping phosphorylation sites. However, mass spectrometry has significant limitations that must be considered when planning to carry out phosphorylation-site mapping. Here we provide an overview of key information that should be taken into consideration before beginning phosphorylation-site analysis, as well as a step-by-step guide for carrying out successful experiments.

FIGURE 1:

Tandem mass spectrometry (MS/MS) analysis of protein phosphorylation. (A) Protein samples are digested with a proteolytic enzyme. The resulting peptides are separated by reverse-phase high-performance liquid chromatography. Peptides enter the mass spectrometer as they elute from the column. Peptide matching is done algorithmically using spectral data and sequence database information. (B) Two basic types of information are generated in the mass spectrometer. The masses of intact peptide ions are determined in a full scan (MS or MS1). Peptides are then isolated one at a time, as depicted for the highlighted peak, and fragmented by colliding them with an inert gas. The resultant fragment ions are detected in a MS/MS (or MS2) scan. With sufficient coverage of fragment ions, the position of the phosphorylated residue, circled in orange, can be identified from the MS/MS (upper path). Fragmentation, however, often liberates the relatively labile phosphate groups at the expense of more informative fragmentation of the peptide backbone (lower path). In extreme cases, MS/MS spectra are dominated by a single ion representing the intact peptide stripped of phosphate. (C) Site localization is dependent on the detection of ions that can distinguish between possible phosphorylatable residues. In the example shown, a threonine and a tyrosine are separated by one amino acid. Fragment ions in the central panel either do not contain a phosphorylatable residue or will have equivalent mass for both possible phosphopeptides. If the threonine is phosphorylated, we would expect to see some or all of the top four site-specific fragment ions shown on the right. If, instead, the phosphate lies on the tyrosine, we would see the bottom four.

FIGURE 2:

Stable-isotope labeling methods for quantitative mass spectrometry. (A) Cells can be labeled either metabolically by growing them in media containing heavy isotope–enriched nutrients such as amino acids in SILAC or chemically after lysis and digestion. Once labeled, samples can be combined for LC-MS/MS analysis. (B) Same sequence peptides from samples labeled with heavy and light isotopes are chemically identical. They coelute from the reverse-phase HPLC column and enter the mass spectrometer together. In a full scan of intact peptides they appear as doublets, separated by the characteristic added mass of the isotope label. Peak heights provide relative quantification. (C) Isobaric labels, such as the depicted 6-plex TMTs, allow multiplexed quantitative analysis. They are chemically incorporated after peptide digestion, before mixing and analysis. (D) The different labels in each set of isobaric labels have identical masses, and thus in the full scan, each peak is actually a composite of peptides from each sample. However, upon fragmentation, each label releases a unique reporter ion that can be detected in a MS/MS scan. Peak heights provide relative quantification of all six samples.