Dataset Information

Phosphoproteome Analysis of Formalin-Fixed and Paraffin-Embedded Tissue Sections Mounted on Microscope Slides

ABSTRACT: Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC–MS sensitivity by miniaturizing nanoLC columns to 25 μm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 μg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD004604 | JPOST Repository | Wed Jul 20 00:00:00 BST 2016

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	110630gen-06FFPE5s-1.mgf	Mgf
	110630gen-06FFPE5s-1.wiff	Wiff
	110630gen-07FFPE5s-2.mgf	Mgf
	110630gen-07FFPE5s-2.wiff	Wiff
	110709gen-04FFPE5s1-01.mgf	Mgf

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Publications

Phosphoproteome analysis of formalin-fixed and paraffin-embedded tissue sections mounted on microscope slides.

Wakabayashi Masaki M Yoshihara Hiroki H Masuda Takeshi T Tsukahara Mai M Sugiyama Naoyuki N Ishihama Yasushi Y

Journal of proteome research 20131223 2

Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-tran ...[more]

PMID: 24328109

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