Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot

Summary

Detailed protocol and three Python scripts are provided for operating an open-source robotic liquid handling system to perform semi-automated protein sample preparation for mass spectrometry experiments, covering detergent removal, protein digestion, and peptide desalting steps.

Abstract

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, which can take up significant person-hours of bench labor and present a source of batch-to-batch variability. Lab automation with pipetting robots can reduce manual work, maximize throughput, and increase research reproducibility. Still, the steep starting prices of standard automation stations make them unaffordable for many academic laboratories. This article describes a proteomics sample preparation workflow using an affordable, open-source automation system (The Opentrons OT-2), including instructions for setting up semi-automated protein reduction, alkylation, digestion, and clean-up steps; as well as accompanying open-source Python scripts to program the OT-2 system through its application programming interface.

Introduction

Mass spectrometry-based shotgun proteomics is a powerful tool to measure the abundance of many proteins in biological samples simultaneously. Proteomics experiments with bioinformatics analysis are routinely employed to identify biomarkers and discover associated biological complexes and pathways underpinning pathological mechanisms. With its high analyte specificity and potential quantitative accuracy, shotgun proteomics also has excellent potential to be adopted by research facilities and diagnostic laboratories for clinical sample analysis without the need to rely on antibodies^1,2.

Protocol

The developed Python scripts have been deposited on GitHub at: https://github.com/MaggieLam-Lab/StandardDigestion-Opentrons. A copy of the scripts is given in Supplementary File 1. Please refer to the GitHub repository for the latest versions.

1. Experimental preparations

1. Check the required hardware before starting the protocol.
   ​NOTE: The following hardware components are required: OT-2 pipettes, pipette tips, 4-in-1 tube rack set, aluminum block set, magnetic module, temperature module, 96-well 2 mL deep well plates (see Table of Materials).

Results

Three Python scripts are provided here that are compatible with the OT-2 robot, and that perform sample preparation for mass spectrometry proteomics with a single protein standard bovine serum albumin (technical replicates n = 5 digestions) and a detergent-containing human heart lysate sample (n = 5 digestions). Each digest product is partitioned into two peptide clean-up reactions. The number of identified peptide-spectrum matches (PSMs), peptides, and proteins in each run of the BSA and heart samples are shown in

Discussion

Critical steps within the protocol
For the best performance, Opentrons-verified labware, modules, and consumables compatible with OT-2 should be used. Custom labware can be created following Opentrons' instruction at Reference¹⁴. Make sure to calibrate the OT-2 deck, pipettes, and labware when used for the first time. It is also critical to follow guidelines from SP3 beads' manufacturer to prepare beads for peptide and protein clean-up. Notably, during the bead .......

Materials

Name	Company	Catalog Number	Comments
300 µL pipette tips	Opentrons
4-in-1 tube rack set	Opentrons		Each set includes 2 base stands and 4 tube holder tops 1.5mL, 2mL, 15mL + 50mL, 15mL, and 50mL. We use 2mL and 15 mL + 50 mL tops in this study.
Acclaim PepMap 100 C18 HPLC Column	Thermo Scientific	#164568	3 μm particle; 100 Å pore; 75 μm x 150 mm
Acetonitrile LC-MS grade	VWR	#JT9829
Aluminum block set	Opentrons		This block set includes 3 tops that are compatible with 96-well, 2.0 mL tubes and a PCR strip to use with the OT-2 temperature module. We use the 2.0mL tube holder in this manuscript.
Ammonium Bicarbonate	Sigma-Aldrich	# A6141
Bovine Serum Albumin Standard, 2 mg/mL	Thermo Scientific	#23210
Dimethylsulfoxide (DMSO) LC-MS grade	Thermo Scientific	#85190
Dithiothreitol	Sigma-Aldrich	#D5545
EASY-Spray HPLC Columns	Thermo Scientific	#ES800A
EasynLC 1200 Nano LC	Thermo Scientific	#LC140
Ethanol Proof 195-200	Fisher	#04-355-720
Formic Acid LC-MS grade	Thermo Scientific	#85178
Human heart lysate	Novus Biologicals	NB820-59217
Iodoacetamide	Sigma-Aldrich	#I1149
Magnetic tube rack	Thermo Scientific	#MR02
MAXQuant v.1.6.10.43			Tyanova et al., 2016 (https://www.maxquant.org/)
mySPIN 6 Mini Centrifuge	Thermo Scientific	#75004061	benchtop mini centrifuge for quick spin
NEST 2 mL 96-Well Deep Well Plate, V Bottom	Opentrons
OT-2 magnetic module	Opentrons		GEN1
OT-2 P300 single channel pipette	Opentrons		GEN1
OT-2 P50 single channel pipette	Opentrons		GEN1
OT-2 robot pipetting robot	Opentrons	OT-2
OT-2 temperature module	Opentrons		GEN1
Pierce Quantitative Colorimetric Peptide Assay	Thermo Scientific	#23275
Protein LoBind tubes 2.0 mL	Eppendorf	#022431102
Protein Sequence Database	UniProt/SwissProt		https://www.uniprot.org/uniprot/?query=proteome:UP000005640% 20reviewed:yes
Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles, Hydrophobic	Cytiva	#65152105050250
Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles, Hydrophylic	Cytiva	#45152105050250
SpeedVac	Thermo Scientific		Vacuum evaporator
Thermo Q Exactive HF Mass Spectrometer	Thermo Scientific	#IQLAAEGAAPFALGMBFZ
Trypsin MS Grade	Thermo Scientific	#90057
Water LC-MS grade	VWR	#BDH83645.400