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 the proteome is more complex and dynamic than the transcriptome¹⁰. Given these challenges, a large number of mixed populations of cells (i.e., bulk cells) are generally used to generate comprehensive proteome data^11,12,13. However, such bulk measurements average out stochastic variations of individual cells, thus obscure important cell-to-cell variability (i.e., cell heterogeneity)^4,14. Limitations of such bulk measurements become even more severe when the cells of interest only account for a small portion of the total populations of cells (e.g., cancer stem cells within tumors at an early-stage cancer). Therefore, there is a huge knowledge gap between single-cell proteomics and genomics (transcriptomics).

Antibody-based immunoassays (e.g., flow or mass cytometry) are predominantly used for targeted proteomic analysis of single cells^{6,7,15,16,17,18}. However, they suffer from low multiplex, limited specificity, and unavailability of antibodies for new proteins of interest. Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative for accurate protein quantification because of its being antibody-free, high multiplex (≥150 proteins in a single analysis¹⁹), high quantitation accuracy (absolute amounts or concentrations), and high specificity and reproducibility (≤10% CV)^20,21,22,23. Recent significant progress in sample preparation has made MS-based single-cell proteomics possible for quantitative analysis of highly abundant proteins from single human cells. However, MS-based single-cell proteomics is still at the early infancy stage. For example, the most advanced MS platform coupled with ultralow-flow RPLC flow rates can only allow label-free MS detection and quantification of ~670-870 proteins out of the total ≥12,000 proteins in single HeLa cells^24,25.

Currently, there are six MS-based single-cell proteomics approaches available for analysis of single mammalian cells, in which four are for global proteomics (nanoPOTS: nanowell-based Preparation in One pot for Trace Samples²⁶; iPAD-1: integrated proteome analysis device for single-cell analysis²⁷; OAD (oil-air-droplet) chip-based single cell proteomic analysis²⁸; SCoPE-MS: single cell proteomics by mass spectrometry²⁹) and the other two are for targeted proteomics (cLC-SRM: carrier-assisted liquid chromatography (LC) – selected reaction monitoring (SRM)³⁰; cPRISM-SRM: carrier-assisted high pressure, high-resolution separations with intelligent selection and multiplexing coupled to SRM³¹). However, all these approaches have technical drawbacks. nanoPOTS, iPAD-1, and OAD downscale sample processing volume to 2-200 nL and are not ready for broad benchtop applications^26,27,28. For SCoPE-MS, a TMT (tandem mass tag) carrier is added after single-cell processing, so it cannot effectively prevent surface adsorption losses during sample processing when a single tube is used for single-cell processing²⁹, resulting in low reproducibility with a correlation coefficient of only ~0.2-0.4 between replicates³². For cLC-SRM and cPRISM-SRM, using exogenous proteins as a carrier is more suitable for targeted proteomics because peptides from excessive exogenous proteins are frequently sequenced by MS/MS, which greatly reduces the chance for sequencing low abundant endogenous peptides^30,31. Unlike global proteomics for relative quantification, the two targeted proteomics approaches can provide accurate or absolute protein analysis of small numbers of cells with high reproducibility using heavy isotope-labeled internal standards at known concentrations. When compared to cPRISM-SRM that requires prior high-resolution PRISM fractionation, resulting in many fraction samples that need to be analyzed, cLC-SRM has a significant advantage in sample throughput without fractionation and can simultaneously quantify hundreds of proteins in a single analysis but with relatively lower detection sensitivity³⁰. Therefore, cLC-SRM is more accessible and should have broader utilities for accurate multiplexed protein analysis of small numbers of cells as well as mass-limited samples.

Herein we describe a detailed protocol to perform cLC-SRM for convenient targeted proteomics analysis of small numbers of human cells, including single cells. The protocol consists of the following major steps: cell sorting by FACS (fluorescence activated cell sorting), cell lysis and digestion processed in low-volume single polymerase chain reaction (PCR) tubes, LC-SRM data collection, and SRM data analysis using publicly available Skyline software (Figure 1). Its broad utility was demonstrated along with our previously well-established SRM assays by absolute targeted quantification of EGFR/MAPK pathway proteins in 1-100 MCF7 or MCF10A cells and determination of pathway protein copies per cell at a wide dynamic range of concentrations³⁰. We anticipate that with the detailed protocol most proteomics researchers can readily implement cLC-SRM in their laboratories for accurate protein analysis of ultrasmall samples (e.g., rare tumor cells) to meet their project needs.

Figure 1: Overview of all steps in cLC-SRM (carried-assisted one-pot sample preparation coupled with liquid chromatography –selected reaction monitoring). Nonhuman cell lysate digests (e.g., Shewanella oneidensis) are used to pretreat PCR tubes for coating tube surface. Small numbers of human cells or single cells sorted by FACS are collected into pretreated PCR tubes. BSA protein (or nonhuman cell lysate proteome) carrier, heavy internal standard (IS), and TFE (or DDM) are added into sample tubes sequentially for facilitating cell lysis and reducing surface adsorption losses. Conceptually, the combined DDM and nonhuman cell lysate proteome carrier will work well for cLC-SRM. Cell lysis is conducted by sonication, and protein denaturation is achieved by heating at high temperature. DTT and IAA reagents are used for reduction and alkylation, respectively (this step is optional). Trypsin is added for digestion with much higher ratios of trypsin over protein amount than that for standard trypsin digestion. The cap of sample tube is removed and then PCR tube is inserted into LC vial for direct LC-SRM analysis. Collected SRM data are analyzed by using publicly available Skyline software. Please click here to view a larger version of this figure.

Protocol

NOTE: The step-by-step cLC-SRM analysis is shown in Figure 1. 1. Pretreatment of PCR tubes 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. 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. Store coated PCR t…

Representative Results

Small amounts of MCF7 cell lysates (0.5-20 ng equivalent to 5-200 cells) were first used to evaluate the performance of cLC-SRM by targeted quantification of EGFR/MAPK pathway proteins because they are more uniform with less variations when compared to small numbers of cells sorted by FACS. As shown in Figure 2A, XICs clearly shows detection of SRM transitions for ATADDELSFK derived from GRB2 present at ~220,000 copies per MCF7 cell34….

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

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