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

Reputable Mentor II
Reputable Mentor II
Böhm G, Prefot P, Jung S, Selzer S, Mitra V, Britton D, Kuhn K, Pike I, Thompson AH.
J Proteome Res. 2015 May 4.
We present a novel Tandem Mass Tag Solid Phase Amino Labelling (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 labelling with TMTs does require some modification to typical labelling conditions particularly pH. It has been found that complete labelling equivalent to standard basic pH solution phase labelling for small and large samples can be achieved on C18 resins using slightly acidic buffer conditions. Improved labelling behaviour on C18 compared to standard basic pH solution phase labelling is demonstrated. We analysed our samples for histidine, serine, threonine and tyrosine-labelling to determine the degree of over-labelling and observed higher than expected levels (25% of all Peptide Spectral Matches (PSMs)) of over-labelling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution phase labelling protocol. Over-labelling 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-labelling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT-labelling compared to label-free methods. Our results also highlight the importance of searching data for over-labelling when labelling methods are used.
Proteome Sciences R&D GmbH & Co., Proteome Sciences Plc.
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