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Biochemistry

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

Published: November 6th, 2020

DOI:

10.3791/61797

1Biological Sciences Division, Pacific Northwest National Laboratory, 2NHC Key Laboratory of Cancer Proteomics, Department of Oncology, Xiangya Hospital, Central South University, 3Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 4Department of Critical Care Medicine, The First Affiliated Hospital of Nanchang University

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.

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.

Recent technological advances in genomics (transcriptomics) allow for comprehensive and precise analysis of the genome (transcriptome) in single cells1,2,3. However, single-cell proteomics technologies are lagging far behind but are just as important as genomics (transcriptomics) technologies4,5,6,7,8. Furthermore, protein abundance cannot necessarily be inferred from mRNA abundance9, and th....

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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.......

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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........

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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.......

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This work was supported by NIH Grant R21CA223715 (TS) and UG3CA256967 (TS and HL). The experimental work described herein was performed in the Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, a national scientific user facility sponsored by the United States of America Department of Energy under Contract DE-AC05-76RL0 1830.

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NameCompanyCatalog NumberComments
2 mL glass LC vialMicrosolv 9502S-WCVVessel to hold PCR tube for autosample injection
BSASigma-AldrichP0834-10×1mLCarrier protein for greatly reducing surface adsorption losses
DTTThermo ScientificA39255Reagent for reduction
Formic acidThermo Scientific28905Reagent for stopping enzyme reaction
IAAThermo ScientificA39271Reagent for alkylation
Peptide internal standardsNew England peptideTargeted quantification of EGFR/MAPK pathway proteins
RT-PCR tube GeneMate BioexpressT-3035-10.2 mL PCR tube for one-pot sample preparation 
Skyline softwareUniversity of WashingtonPublicly available for SRM data analysis
SonicatorHielscher Ultrasound TechnologyUTR200Sonication on ice for cell lysis
Speed Vac concentratorThermo ScientificReduction of the percentage of TFE for effective trypsin digestion
TFESigma-Aldrich18370-10×1mL60% TFE for cell lysis
Thermocycler w/ heated lidPeltier Thermal Cycler PTC-200Heating for protein denaturation
Trypsin GoldPromegaV528AEnzyme for protein digestion 
Waters BEH C18 columnWatersC18 column for peptide separation

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