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Peptide probes containing a non-hydrolyzable phosphotyrosine-mimetic residue as a tool for tyrosine phosphatomics

ABSTRACT: Protein tyrosine phosphatases (PTPs) cooperate with protein tyrosine kinases to regulate intracellular tyrosine phosphorylation levels, and their dysfunction is associated with various diseases. However, due to the low abundance of PTPs in cells, comprehensive analysis requires an enrichment step prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS). In this study, we designed and synthesized peptide probes for PTP pulldown assay using a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F2Pmp). We found that different F2Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F2Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F2Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD025038 | JPOST Repository | Wed Oct 27 00:00:00 BST 2021

REPOSITORIES: jPOST

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Peptide probes containing a non-hydrolyzable phosphotyrosine-mimetic residue for enrichment of protein tyrosine phosphatases.

Tsumagari Kazuya K Niinae Tomoya T Otaka Akira A Ishihama Yasushi Y

Proteomics 20211221 4

We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F2 Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F2 Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F2 Pmp failed to enrich PT ...[more]

PMID: 34714599

Similar Datasets

Project description:Signal propagation mediating numerous cellular processed in T cells is orchestrated by tyrosine phosphorylation with a high degree of regulation1, 2. Because aberrant tyrosine phosphorylation can result in many diseases3, a method to systematically quantify this dynamic process can be helpful in understanding the mechanistic underpinnings of T cell functions. However, accurate quantitation of tyrosine phosphorylation is challenging due to its low abundance4. Mass spectrometry(MS) based phosphotyrosine proteomics has emerged to be an attractive solution for wide scale profiling of the tyrosine phosphoproteome5, 6. Many advances have been made in this field to improve the enrichment efficiency of phosphotyrosine(pTyr)-containing peptides to improve the coverage depth7-11. Separately, computational methods of Fourier transform-(FT)MS such as an Orbitrap analyzer have also made significantly progress in the quantitation of TMT samples. For example, the phase-constrained spectrum deconvolution method (ΦSDM) have been shown to substantially improve the spectral quality and speed of FT-MS instruments12. ΦSDM deconvolves FT spectra into frequency distributions to allow more efficient extraction of the harmonic components of the oscillating ions12. As a result, higher mass resolution and accuracy can be achieved with shorter transient times12. We recently developed the BOOST approach by coupling PV boost channel in a multiplexed TMT approach to significantly increase the quantitative depth of the tyrosine phosphoproteome13. While it has been shown to work in Jurkat cell line, it has not been demonstrated to work in scarce mice primary T cells. Here, we demonstrate the first phosphotyrosine proteomics performed in mice primary T cells using BOOST that enabled the identification of more than 6000 pTyr peptides using only 1 mg of protein. We further demonstrated the importance of ΦSDM, in which when disabled, enabled significantly deeper quantitation depth in low-abundance samples. Using samples with contrived ratios, disabling ΦSDM improved the precision of low-abundance peptides and allowed the quantitation of up to 2-fold more increase in statistically significant ratios.

			Action	DRS
	180718kt_02PTP_2_c1.raw	Raw
	180718kt_04PTP_2_c2.raw	Raw
	180718kt_06PTP_2_p1.raw	Raw
	180718kt_08SH2_3_p1.raw	Raw
	180725kt_02PTP_2_p2.raw	Raw