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

J Proteome Res. 2015 Jun 5;14(6):2500-10. doi: 10.1021/acs.jproteome.5b00072. Epub 2015 May 13.


We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h 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 standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling 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. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.

Keywords: Mass spectrometry; TMT; chemical labeling; isobaric tags; phosphorylation; proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amines / chemistry
  • Cell Line, Tumor
  • Humans
  • Hydrogen-Ion Concentration*
  • Phosphopeptides / chemistry*
  • Tandem Mass Spectrometry


  • Amines
  • Phosphopeptides