PXD002072

PXD002072 is an original dataset announced via ProteomeXchange.

Title	Low pH Solid Phase Amino-Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis
Description	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.
HostingRepository	PRIDE
AnnounceDate	2015-04-21
AnnouncementXML	Submission_2015-04-21_02:49:11.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Vikram Mitra
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	deamidated residue; monohydroxylated residue; phosphorylated residue; iodoacetamide derivatized residue; TMT6plex-126 reporter+balance reagent acylated residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2015-04-15 08:15:45	ID requested
⏵ 1	2015-04-21 02:49:12	announced

Dataset with its publication pending

curator keyword: Technical

submitter keyword: TMT, Low pH Solid Phase Amino-Labeling,SPAL, SysQuant

Andrew H. Thompson
contact affiliation	Proteome Sciences Plc, Coveham House, Downside Bridge Road, Cobham KT11 3E, UK
contact email	andrew.thompson@proteomics.com
lab head
Vikram Mitra
contact affiliation	Proteome Sciences
contact email	vikram.mitra@proteomics.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD002072
     1. Label: PRIDE project
     2. Name: Low pH Solid Phase Amino-Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis