Phosphoproteomics for the masses

Abstract

Protein phosphorylation serves as a primary mechanism of signal transduction in the cells of biological organisms. Technical advancements over the last several years in mass spectrometry now allow for the large-scale identification and quantitation of in vivo phosphorylation at unprecedented levels. These developments have occurred in the areas of sample preparation, instrumentation, quantitative methodology, and informatics so that today, 10 000-20 000 phosphorylation sites can be identified and quantified within a few weeks. With the rapid development and widespread availability of such data, its translation into biological insight and knowledge is a current obstacle. Here we present an overview of how this technology came to be and is currently applied, as well as future challenges for the field.

Figure 1. Cell-cycle-regulated phosphorylation of the kinome

(A) Protein kinase networks in mitosis are depicted within the context of the human kinome, represented as a dendrogram. Protein kinases for which the identified phosphopeptides were more than 4-fold up-regulated in M phase and contain consensus phosphorylation sites for CDK, PLK, or Aurora kinases are included. (B) An alignment of kinases having activation loops which contained phosphorylation sites that changed in abundance with progression of the cell cycle. The identified phosphopeptides and phosphorylation sites are indicated with yellow highlighting and red lettering, respectively. The ratios of relative abundances in M and S phases (M/S) observed are indicated. M/S ratios which could not be normalized for protein expression are marked by an asterisk (*). All panels reprinted with permission from (21), with the left panel originally adapted from www.cellsignaling.com.

Figure 2. Phosphopeptide enrichment

The first challenge in phosphoproteomics is enrichment of low-abundance phosphopeptides or phosphoproteins. The most commonly used enrichment techniques exploit the chemical characteristics of the phosphate group in affinity capture. The figure panels have been modified with permission from previous publications (–125).

Figure 3. Phosphopeptide fragmentation

For all panels, ETD results are shown in blue and CAD in red. (A, C) The probability of a high confidence phosphopeptide identification via either ETD or CAD MS/MS for peptide cations having various charge (z) as a function of precursor m/z ratio is indicated (65). To evaluate the importance of any one m/z ratio bin, the percentage of all precursors observed having the specific z and m/z ratio are given below. Note, that for dications, CAD is the most successful method; however, for triply charge cations, ETD is the best method for peptides below 750 m/z. (B) A probabilistic decision tree for using ETD and CAD together for phosphopeptide sequencing was generated from the data represented in panels A and C, as well as those for other charge states indicated. (D–G) Comparison of CAD and ETD tandem mass spectra for representative doubly and triply-charged phosphopeptides. (H–J) The percentage of all backbone bonds cleaved via ETD or CAD for the 3 backbone bonds to the N-terminal side (−3 through −1) and the C-terminal side (+1 through +3) of a phosphorylated serine (H), threonine (I) or tyrosine (J) (13). Panels A–C from the Supporting Information from (65) and panels H–J reprinted with permission from (13).

Figure 4. Isotope labeling strategies for phosphopeptide quantitation

Metabolic labeling introduces heavy isotopes into proteins synthesized in living cells, using heavy isotope-containing amino acids (SILAC) or nutrients (¹⁵N or ¹³C). Peptides analyzed by MS exhibit an m/z difference between light- and heavy-labeled peptides, allowing for relative quantitation by monitoring extracted ion chromatograms of eluting peptides. Isobaric tagging strategies (e.g., iTRAQ and TMT) label peptides from different samples after protein digestion is performed. As co-eluting peptides modified with tags having the same nominal mass are isolated and fragmented, the MS² scan provides assessment of relative abundance through analysis of the intensity of low m/z reporters. Isotope tagging strategies label digested peptides with heavy and light reactive tags (e.g., ICAT), which allow for determining relative abundance from extracted ion chromatograms. Absolute quantitation (AQUA) of phosphopeptides can be achieved using isotope dilution, in which a known amount of a synthetic heavy isotope-labeled phosphopeptide is spiked into a sample after enzymatic digestion is performed to produce the corresponding endogenous phosphopeptide of interest. Quantitation is achieved by selected reaction monitoring (SRM), typically performed on a triple quadrupole mass spectrometer. For all strategies, the step in the workflow which incorporates heavy isotopes is indicated, with blue representing samples with naturally occurring light isotopes and red representing samples containing stable heavy isotopes. Figure panels adapted with permission from the following references (96, 126).