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Challenges in Single Cell ProteomicsVirginie Sjoelund, PhD
Laboratory for Molecular Biology and Cellular Research
University of Oklahoma Health Sciences Center, OKC, OK
The Promise Behind Single Cell Applications
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Methodology
1. Single cell isolation2. Cell lysis3. Digestion4. Label free or TMT labeling?5. Desalting6. Mass Spectrometry7. Analysis
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# Pubmed publications containing term “single cell proteomics”
Single Cell Isolation
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The FACS core will become your best friend!
Should be doing routinely in 96 or 384 well plates.Exclude dead cells and debrisCan select cells of similar size to simplify analysis
Other option: Sort on NanoPOT slide (need to fabricate it)
NanoPOT slide
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Zhu et al. Nat Commun. 2018 Feb 28;9(1):882
1mm
2.25mm
5x13 nano wells Retains hydrophilicity
Treated with 2% PFDS in 2,2,4-trimethylpentane
Affixed with silicone adhesive
30µm layer of PDMS
PFDS: (heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane
2,2,4-trimethylpentane
PDMS:Polydimethylsiloxane
Cell Lysis
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1. Boiling followed by sonicationDirectly after sorting, plates are quickly spun and boiled for 5min at 95oCFollowed by sonication for 2min in waterbath sonicator to finish lysis
2. Freeze thawing cyclesFreeze at -80oC for at least 5min and heat at 90oC for at least 10min
Digestion
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Regular trypsin digest
TMT Labeling
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Need carrier cells to boost signal (20 to 200 cells)Need empty label to check S/N ratio
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TMT10-126
TMT10-127N
TMT10-127C
TMT10-128N
TMT10-128C
TMT10-129N
TMT10-129C
TMT10-130N
TMT10-130C
TMT10-131
TMT11-131
1 Cell
1 Cell
1 Cell
1 Cell
1 Cell
1 Cell
1 Cell
1 Cell
1 Cell
100 Cells
Abun
danc
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TMT Channel
1 cellEmpty
Booster
Desalting
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Peptides are either loaded onto a capillary and then desalted on a trap columnPeptides are dried in speedvac and then loaded onto a trap column for washing
Cong et al. Anal Chem. 2020 Jan 21
Mass Spectrometry
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Instruments used: Thermo Orbitrap Fusion LumosThermo Orbitrap EclipseThermo Orbitrap Exploris
Importance of FAIMSImportance of smaller ID column
Analysis
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Proteome Discoverer MaxQuant
Importance of Real-time search
TMT Results
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Label-Free, Orbitrap Tribrid Eclipse
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Label-Free, Orbitrap Tribrid Eclipse + FAIMS
Protein Groups
Eclipse Eclipse +FAIMS Gain
1 HeLa Cell 551 829 50%
100 HeLa Cells 2109 3067 45%
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Label-Free, Orbitrap Exploris
Smaller ID column (30µmx30cm, 1.7µm-CoAnn Tech) at lower nano flow rate (50nL/min – Thermo Ultimate 3000 RSLCnano System) provides high performance with high sensitivity
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Single HeLa Cell 1.0ng HeLa Digest 2.0ng HeLa Digest
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Single HeLa Cell 1.0ng HeLa Digest 2.0ng HeLa Digest
-FAIMS +FAIMS
Pept
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LFQ LFQ LFQ TMT TMT TMT
Sample Prep NanoPots NanoPots NanoPots NanoPots NanoPots Plate
Separation 30um i.d. Column 20um i.d. Column 30um i.d. Column 30um i.d. Column 30um i.d. Column 75um i.d. Column
Mass Spec Lumos Eclipse + FAIMS Exploris Lumos Eclipse + RT search Elite
Data Analysis MQ PD PD PD PD MQ
Protein Groups 211 829 741 1676 2346 767
Year 2018 2019 2019 2019 2020 2018
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Lumos Exploris Elite Eclipse +FAIMS
Lumos Eclipse + RTsearch
Red: 2018Blue: 2019Black: 2020
Circle: LFQSquare: TMT
