Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

A comparison of cartridge and bead-based sample preparation strategies for bottom-up proteome analysis of detergent-containing samples (#708)

Jessica L. Nickerson 1 , Kent MacLean 2 , Alan A. Doucette 1
  1. Chemistry, Dalhousie University, Halifax, NS, Canada
  2. Proteoform Scientific, Halifax, NS, Canada

Introduction

Detergents including sodium dodecyl sulfate (SDS) are becoming increasingly accepted in proteomics workflows. This is owing to a growing list of semi-automated approaches, designed to effectively purify and digest protein mixtures ahead of bottom up LC-MS/MS analysis. Since the advent of filter-aided sample preparation (FASP) as a cartridge-based format which simplifies SDS removal and protein digestion, numerous other strategies have been reported enabling the MS acquisition of the resulting peptide digests.1 Among the cartridge and bead-based technologies are Suspension Trapping S-Trap, in-StageTip (iST), Single-Pot, Solid-Phase-Enhanced Sample Preparation (SP3), and the ProTrap XG, all of whom report successful acquisition of peptide lists from SDS-containing samples.1,2,3,4,5Here, we directly evaluate the performance of these sample preparation strategies looking at four specific figures of merit: the protein/ peptide recovery, sample purity as judged by residual level of SDS, protein digestion efficiency, and sample throughput/ process time.

 

Methods

Sample preparation will be conducted within FASP, S-Trap, iST, SP3 and ProTrap XG, based on the respective user manuals. Test proteome mixtures will be prepared in SDS at varying concentrations of protein to assess the effectiveness of the sample preparation at different sample loads. Protein recovery will be quantified by the bicinchoninic acid (BCA) assay, as well as SDS PAGE, and LC-UV analysis. Purity will be assessed by quantifying residual SDS using the methylene blue active substances assay. The resulting peptide mixtures will be subject to bottom up LC-MS/MS analysis, assessing both the number of peptides, degree of missed cleavage, and coverage of the proteome with respect to intrinsic protein properties (molecular weight, hydrophobicity).

 

Results

Methods are in place to evaluate performance (purity/ yield/ digestion efficiency) of each sample preparation strategy.

 

Conclusions

Multiple strategies exist for bottom-up MS analysis of detergent-containing samples. This study provides unbiased assessment of the performance characteristics of several popular strategies.

  1. 1. Mann et al. (2007). Nature Protocols, 2, 1896-1906.
  2. 2. Mann et al. (2009). Nature Methods, 6, 359-62.
  3. 3. Krijgsveld et al. (2014). Molecular Systems Biology, 10.
  4. 4. Yu et al. (2018). Journal of Proteome Research, 17, 2917-2924.
  5. 5. Doucette et al. (2015). Journal of Proteomics, 118, 140-150.