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Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

ABSTRACT: Label-free quantification is a powerful method for studying cellular protein phosphorylation dynamics. However, whether current data normalization methods achieve sufficient accuracy has not been examined systematically. Here, we demonstrate that a large uni-directional shift in the phosphopeptide abundance distribution is problematic for global median centering and quantile-based normalizations and may mislead the biological conclusion from unlabeled phosphoproteome data. Instead, we present a novel normalization strategy, named pairwise normalization, which is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of pairwise normalization was validated by statistical methods, western blotting analysis, and by bioinformatics analysis. In addition, we demonstrate that the choice of normalization method influences the downstream analyses of the data and perceived pathway activities. Furthermore, we demonstrate that the developed normalization method, combined with pathway analysis algorithms, revealed a novel biological synergism between Ras signalling and PP2A inhibition by CIP2A.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Susumu Imanishi

LAB HEAD: Susumu Y. Imanishi

PROVIDER: PXD001374 | Pride | 2015-08-24

REPOSITORIES: Pride

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			Action	DRS
	130317_26_SCR_X5_TiO2_45_10MS2_110min.raw	Raw
	130317_26_SCR_X5_TiO2_45_10MS2_110min_Mascot.msf	Msf
	130317_26_SCR_X5_TiO2_45_10MS2_110min_SpectraST.msf	Msf
	130317_28_SCR_Y5_TiO2_45_10MS2_110min.raw	Raw
	130317_28_SCR_Y5_TiO2_45_10MS2_110min_Mascot.msf	Msf

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Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling.

Kauko Otto O Laajala Teemu Daniel TD Jumppanen Mikael M Hintsanen Petteri P Suni Veronika V Haapaniemi Pekka P Corthals Garry G Aittokallio Tero T Westermarck Jukka J Imanishi Susumu Y SY

Scientific reports 20150817

Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification ...[more]

PMID: 26278961

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