PTM site localization and isomer differentiation of phosphorylated peptides 

Tunable electron activated dissociation (EAD) MS/MS using the SCIEX ZenoTOF 7600 system

Joanna Bons1, Jason Cason2, Birgit Schilling1, Christie Hunter3
1
Buck Institute, USA, 2SCIEX, Canada, 3SCIEX, USA

Abstract

Specific location of phosphorylation sites on proteins is important in fully understanding their role in cellular processes. However, depending on the peptide sequence, this can be challenging. Here, the use and benefits of tunable electron activated dissociation (EAD) for phosphopeptide analysis and confident site-localization was evaluated on the ZenoTOF 7600 system. The combination of Zeno MS/MS with EAD provided high quality MS/MS for full characterization of the peptide including clear differentiation of the two phosphopeptides isomers studied. Fragmentation patterns for the two phosphopeptide isomers were compared between EAD and collision induced dissociation (CID), direct evidence for the location of the phosphorylation on the two peptides was only observed using EAD. Skyline software was used for processing the MRMHR data, both the CID and EAD data.

RUO-MKT-02-13174-A_f0

Introduction

Protein phosphorylation is an important post-translational modification (PTM) as it is involved in a large variety of dynamic cellular processes. However, PTM site localization and quantification of phosphopeptides by collision induced dissociation (CID) MS/MS can be challenging, and phospho-peptides can exhibit a partial neutral loss of the phospho group (‑98 Th). Phospho-isomer differentiation and subsequent precise PTM site localization can be achieved by measuring isomer-specific ions containing the actual modification (direct evidence), or by measuring differentiating fragment ions that do not contain the modification (indirect evidence). Depending on the peptide sequence, detecting near complete fragment ion series, and more particularly the challenging fragment ions that would define the peptide C- and N- termini, can be necessary for PTM-site localization, such as for pS-56 and pS-59 of the NDUFA10 subunit of mitochondrial Complex I1 (Figure 1). The benefits of electron activated dissociation (EAD) versus CID were first explored for malonylation PTM.2

In this study, the use and benefits of EAD fragmentation for phosphopeptide analysis, site-localization and differentiation, and MS/MS-based phosphopeptide quantification were evaluated.

Figure 1. MS/MS spectra of LITVDGNICSGKpSK peptide analyzed with CID and EAD fragmentation. The phosphorylated peptide at m/z 505.58 (z=3) was analyzed in (A) CID mode and (B) EAD mode (kinetic energy, KE = 2). CID fragmentation resulted in low abundant and noisy PTM-specific differentiating ions (y and prominent y-98, no detected b ions). However, comprehensive EAD MS/MS generated distinct z and c fragment ions that provided evidence for definitive PTM site localization.

Key features of EAD for phosphoproteomics

  • Efficient electron activated dissociation (EAD)3 generates strong and distinct PTM site localization ions, enabling phospho-isomer differentiation
  • Tunable kinetic energy (KE) for EAD MS/MS allows for selection of KE that provides the highest fragment ion abundance, while not inducing neutral loss from the phosphoryl group (-98 Th)
  • Generation of strong PTM-containing site localization ions, even small z+1 ions (z2+1, z3+1 and z4+1) and high c ions (c10, c11 and c12)4
  • The optimal KE values are different between different types of modifications, phosphorylation and malonylation2
  • Using the Zeno trap gives up to ~10x increase in intensity for key site-localizing fragment ions (small z+1 ions and high c ions)
  • Preliminary quantification using EAD high-resolution MRM (MRMHR)5 indicates good linearity over concentrations interrogated
  • Detailed characterization and MS/MS-based quantification of the modified peptides analyzed by EAD MS/MS using Skyline.