Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Summary

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

Abstract

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

Introduction

Recent technological advances in genomics (transcriptomics) allow for comprehensive and precise analysis of the genome (transcriptome) in single cells^1,2,3. However, single-cell proteomics technologies are lagging far behind but are just as important as genomics (transcriptomics) technologies^4,5,6,7,8. Furthermore, protein abundance cannot necessarily be inferred from mRNA abundance⁹, and th....

Protocol

NOTE: The step-by-step cLC-SRM analysis is shown in Figure 1.

1. Pretreatment of PCR tubes

1. Add 100 µL of nonhuman (e.g., Shewanella oneidensis) cell lysate digests at 0.2 µg/µL to PCR tubes. Incubate at room temperature for overnight to coat PCR tube surface.
2. Remove the cell lysate digests by pipetting, rinse PCR tubes with HPLC-grade water 3 times, and then airdry PCR tubes in a fume hood.
3. Store coated PCR t.......

Discussion

cLC-SRM is a convenient targeted proteomics method that enables accurate multiplexed protein analysis of small numbers of cells including single cells. This method capitalizes on protein carrier-assisted one-pot sample preparation, in which all steps including cell collection, multistep cell lysis and digestion, and transfer of peptide digests to capillary LC column for MS analysis are performed in one pot (e.g., single tube or single well) (Figure 1). This 'all-in-one' low-volume on.......

Materials

Name	Company	Catalog Number	Comments
2 mL glass LC vial	Microsolv	9502S-WCV	Vessel to hold PCR tube for autosample injection
BSA	Sigma-Aldrich	P0834-10×1mL	Carrier protein for greatly reducing surface adsorption losses
DTT	Thermo Scientific	A39255	Reagent for reduction
Formic acid	Thermo Scientific	28905	Reagent for stopping enzyme reaction
IAA	Thermo Scientific	A39271	Reagent for alkylation
Peptide internal standards	New England peptide		Targeted quantification of EGFR/MAPK pathway proteins
RT-PCR tube	GeneMate Bioexpress	T-3035-1	0.2 mL PCR tube for one-pot sample preparation
Skyline software	University of Washington		Publicly available for SRM data analysis
Sonicator	Hielscher Ultrasound Technology	UTR200	Sonication on ice for cell lysis
Speed Vac concentrator	Thermo Scientific		Reduction of the percentage of TFE for effective trypsin digestion
TFE	Sigma-Aldrich	18370-10×1mL	60% TFE for cell lysis
Thermocycler w/ heated lid	Peltier Thermal Cycler	PTC-200	Heating for protein denaturation
Trypsin Gold	Promega	V528A	Enzyme for protein digestion
Waters BEH C18 column	Waters		C18 column for peptide separation