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Low pH Solid Phase Amino-Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis

ABSTRACT: We present a novel Tandem Mass Tag Solid Phase Amino Labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatised (C18) solid supports. This method can reduce the number of steps required in complex protocols saving time and potentially reducing sample losses. In our global phosphopeptide profiling workflow (SysQuant) we can cut 24 hours from the protocol while increasing peptide identifications (20%) and reducing side-reactions. Solid phase labeling with TMTs does require some modification to typical labeling conditions particularly pH. It has been found that complete labeling equivalent to 2 standard basic pH solution phase labeling for small and large samples can be achieved on C18 resins using slightly acidic buffer conditions. Improved labeling behaviour on C18 compared to standard basic pH solution phase labeling is demonstrated. We analysed our samples for histidine, serine, threonine and tyrosine-labeling to determine the degree of over-labeling and observed higher than expected levels (25% of all Peptide Spectral Matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution phase labeling protocol. Over-labeling at all these sites is greatly reduced (four-fold to 7% of all PSMs) by the low pH conditions used in the TMT-SPAL protocol. Over-labeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT-labeling compared to label-free methods. Our results also highlight the importance of searching data for over-labeling when labeling methods are used.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Vikram Mitra

LAB HEAD: Andrew H. Thompson

PROVIDER: PXD002072 | Pride | 2015-04-21

REPOSITORIES: Pride

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			Action	DRS
	20140210_gib437-1-1_Fr01a.raw	Raw
	20140210_gib437-1-1_Fr02a.raw	Raw
	20140210_gib437-1-1_Fr03a.raw	Raw
	20140210_gib437-1-1_Fr04a.raw	Raw
	20140210_gib437-1-1_Fr05a.raw	Raw

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Low pH Solid Phase Amino-Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis

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