Over 40% more proteins identified using Zeno MS/MS  

Using the ZenoTOF 7600 system, with the OptiFlow ion source and OneOmics suite in the cloud

Alexandra Antonoplis1, Bradley Schneider2, Christie Hunter1
1
SCIEX, USA, 2SCIEX, Canada

Abstract

Proteomics samples are some of the most complex samples analyzed by mass spectrometry, therefore the ability to collect high quality MS/MS at very high acquisition rates is key to achieving high numbers of peptide and protein identifications.  The ZenoTOF 7600 system delivers a ~5-fold gain in MS/MS sensitivity when analyzing peptides, so this study investigates the impact of this large sensitivity gain on identification rates. Four different gradient lengths were tested across a range of sample loadings and a series of acquisition parameters were optimized. Over 40% improvements in the number of protein IDs were observed relative to previous state-of-the-art technology for identical sample loadings.

RUO-MKT-02-13174-A_f0

Introduction

Proteomics samples are some of the most complex samples encountered by the mass spectrometry community. With estimates of over a million different proteins and proteoforms existing in the human proteome,1 understanding which proteins are involved in a given biological function or process can be incredibly challenging. Although any one sample alone would never contain all of these proteins, samples can often contain many thousands of different proteins that can span a concentration range of over 10 orders of magnitude.2 The challenge for discovery proteomics techniques is to try to analyze as broadly and as deeply as possible to detect as many of the proteins and proteoforms present so that biological insights can be gained.

Sensitivity (particularly, MS/MS sensitivity) is a key performance specification for proteomics experiments. High sensitivity MS/MS leads to the broad and deep coverage for protein identification (ID). The ZenoTOF 7600 system uses Zeno trap technology to increase duty cycle to ≥90% across the entire fragment ion mass range, resulting in gains of 4-25 fold in MS/MS mode.3 These gains alone would enable large improvements in proteomics discovery experiments.

The data acquisition strategy is another key factor that can influence proteomics experiments. One of the main acquisition workflows for discovery proteomics is data dependent acquisition (DDA), also known as information dependent acquisition (IDA). During data acquisition, the highest ranking ions that pass certain criteria in each cycle are sent for MS/MS analysis. With many factors to consider, an important consideration is how to specify the selection criteria in order to maximize the number of unique, high confidence peptide and protein IDs. 

In the current study, the sensitivity gains using the Zeno trap technology were evaluated for the impact on protein ID results. Additionally, Zeno IDA acquisition parameters were optimized for 4 different microflow gradient lengths using a design of experiments (DOE) approach. 

Figure 1. Increase in peptide identifications with the ZenoTOF 7600 system. Peptide Identifications at 1% global false discovery rate (FDR) are plotted in blue for the ZenoTOF system and compared to the TripleTOF 6600 system (red). A 10 min gradient length was used and the TripleTOF system values are an average from three different instruments.4

Key features of the ZenoTOF 7600 system for protein identification

  • The ZenoTOF 7600 system delivers a 4 to 25-fold gain in MS/MS sensitivity leading to many more high confidence peptide and protein IDs
  • Optimized IDA workflows using DOE were determined for a range of microflow gradient lengths by comparing the number of peptide and protein IDs at 1% global false discovery rate
  • Microflow chromatography and the OptiFlow source provide both robustness and ease-of-use for reliable low-flow liquid chromatography
  • Cloud-based ProteinPilot app in the OneOmics suite enables fast and seamless data processing with many features for deriving biological insights

Â