pubmed.ncbi.nlm.nih.gov

Motif-specific sampling of phosphoproteomes - PubMed

Motif-specific sampling of phosphoproteomes

Cristian I Ruse et al. J Proteome Res. 2008 May.

Abstract

Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Solution pH is initially adjusted to 3.5 and upon addition of Ba2+/acetone, a first set of phosphopeptides is precipitated. Dried supernatant is solubilized and adjusted to pH 4.6 and precipitated again. The remaining supernatant is dried, solubilized, solution pH adjusted to 8.0 and precipitated again. Individual fractions are independently analyzed using MudPIT. Data were acquired using our MudPIT platform on a linear ion trap mass spectrometer or a hybrid linear ion trap orbitrap mass spectrometer.

Figure 2
Figure 2

Identification of phosphoproteome sampled with different Ba2+ ions concentrations and analyzed MudPIT. A. Enrichment of phosphopeptides per pH fraction for three different Ba2+ molarities. B. Overlapping segments of phosphoproteome precipitated by different Ba2+ molarities: 7.5 µmol (yellow), 6 µmol (red) and 1 µmol (blue).

Figure 2
Figure 2

Identification of phosphoproteome sampled with different Ba2+ ions concentrations and analyzed MudPIT. A. Enrichment of phosphopeptides per pH fraction for three different Ba2+ molarities. B. Overlapping segments of phosphoproteome precipitated by different Ba2+ molarities: 7.5 µmol (yellow), 6 µmol (red) and 1 µmol (blue).

Figure 3
Figure 3

Proportional Venn diagram describing overlap of identified phosphopeptides among pH 3.5, 4.6 and 8.0 fraction.

Figure 4
Figure 4

Sequence logos of phosphopeptides from nuclear extract of HeLa cells: pH 3.5 (A), pH 4.6 (B) and pH 8.0 (C). Sequence logos were built for amino acids lining of Ba2+ ions binding pockets (−5 to +5 AAs). Sequence logos statistically evaluate the binding properties of a population of phosphopeptides by measuring the information (in bits) required at each position around the phosphosite for interaction with barium ions. D. Sequence logo profiles of doubly phosphorylated peptides identified in pH 3.5 Ba2+/acetone fraction of HeLa cell nuclear extract.

Figure 4
Figure 4

Sequence logos of phosphopeptides from nuclear extract of HeLa cells: pH 3.5 (A), pH 4.6 (B) and pH 8.0 (C). Sequence logos were built for amino acids lining of Ba2+ ions binding pockets (−5 to +5 AAs). Sequence logos statistically evaluate the binding properties of a population of phosphopeptides by measuring the information (in bits) required at each position around the phosphosite for interaction with barium ions. D. Sequence logo profiles of doubly phosphorylated peptides identified in pH 3.5 Ba2+/acetone fraction of HeLa cell nuclear extract.

Figure 4
Figure 4

Sequence logos of phosphopeptides from nuclear extract of HeLa cells: pH 3.5 (A), pH 4.6 (B) and pH 8.0 (C). Sequence logos were built for amino acids lining of Ba2+ ions binding pockets (−5 to +5 AAs). Sequence logos statistically evaluate the binding properties of a population of phosphopeptides by measuring the information (in bits) required at each position around the phosphosite for interaction with barium ions. D. Sequence logo profiles of doubly phosphorylated peptides identified in pH 3.5 Ba2+/acetone fraction of HeLa cell nuclear extract.

Figure 5
Figure 5

Sequence logos of phosphopeptides from whole cell lysates of 5minute iso-stimulated HEK cells overexpressing β1AR: pH 3.5 (A), pH 4.6 (B) and pH 8.0 (C). Sequence logos were built for amino acids lining of Ba2+ ions binding pockets (−5 to +5 AAs).

Figure 5
Figure 5

Sequence logos of phosphopeptides from whole cell lysates of 5minute iso-stimulated HEK cells overexpressing β1AR: pH 3.5 (A), pH 4.6 (B) and pH 8.0 (C). Sequence logos were built for amino acids lining of Ba2+ ions binding pockets (−5 to +5 AAs).

Figure 6
Figure 6

Log2 ratio of protein expression levels (2 min stimulation / 30 sec stimulation) and their corresponding phosphorylation levels.

Figure 7
Figure 7

Network centered on MAPK1. Canonical pathways are highlighted. Legend: arrows – proteins that act upon another protein, lines – protein-protein interactions, numbers below shapes – protein expression levels, shapes – inverted triangle/kinase, diamond/enzyme, double circle/group or complex, circle/other. Filled shapes – dataset file genes, Non-filled shapes – Knowledge base genes. Red color represents up-regulation and green color down-regulation. While we kept the original IPA color codes, our majority of the proteins showed unchanged expression levels.

Similar articles

Cited by

References

    1. Hunter T. Signaling--2000 and beyond. Cell. 2000;100:113–127. - PubMed
    1. Loyet KM, Stults JT, Arnott D. Mass spectrometric contributions to the practice of phosphorylation site mapping through 2003: a literature review. Mol Cell Proteomics. 2005;4:235–245. - PubMed
    1. Ficarro SB, et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol. 2002;20:301–305. - PubMed
    1. Beausoleil SA, et al. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A. 2004;101:12130–12135. - PMC - PubMed
    1. Bodenmiller B, Mueller LN, Mueller M, Domon B, Aebersold R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods. 2007;4:231–237. - PubMed

Publication types

MeSH terms

Substances