ChallengeConcept of boosting strategy

Challenge of boosting strategy

(a) isotopic “leakage” issue (b) Ion sampling

Bulk sample

Single cell sample

Real single-cell proteomics#

Chia-Feng Tsai1, Aivett Bilbao2, Sarah M. Williams2, Jongmin Woo2, Kristin Engbrecht1, Ronald J. Moore1, Richard D. Smith1, Ljiljana Pasa-Tolic2, Tao Liu1 , and Ying Zhu2

1Biological Sciences Division and 2Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA

FAIMS-enhanced Boosting-free and Multiplexed Single-cell Proteomics Analysis

Workflow

Isobaric match between run

Figure 1. The BASIL strategy[1]. BASIL uses one of the isobaric tandem mass tag (TMT)channels as the boosting channel (i.e., with much higher sample input) to enhance thedetection of low abundance peptides or phosphopeptides at MS1 level. After MS/MSfragmentation of peptide backbone, the peptide sequence information is obtained withthe major contribution from the boosting sample, while the quantitation signal isrecorded from the TMT reporter ion intensities in individual sample channels.

Figure 2. The challenge of boosting like strategy. (a) The isotopic impurity of theTMT134N reagent used for the boosting channel resulted in the isotopic leakage due tothe isotopic impurity. (b) The spiking of boosting materials decreased the ion samplingefficiency for single-cell samples.

Figure 4. The isobaric match between run procedure. (a) Peptide features in single cells areidentified by matching to the spectral library (Match-Between-Run, MBR) based on three-dimensional tags including retention time, m/z, and FAIMS CVs through MaxQuantsoftware[4]. (b) After MBR, the TMT information in MS/MS scans which has accurate RT,precursor m/z (10 ppm error) and the same FAIMS CV to the matched ion is extracted forquantitation.

Figure 3. Workflow of the TIFF method[2] (Transferring Identification based on FAIMSFiltering). High-input samples (usually from 10 ng peptides with TMT labeling) are analyzedby LC-FAIMS-MS with each LC-MS analysis utilizing a discrete FAIMS CV to generate aspectral library; single-cell samples with TMT labeling are analyzed by cycling throughmultiple FAIMS CVs for each LC-MS analysis.

Reference: 1. Anal. Chem. 91, 5794 (2019)

2. bioRxiv. 2021; 4283333. J. Proteome Res. 19, 10, 3945 (2020)4. bioRxiv. 2021; 431689

www.omics.pnl.govCONTACT: Chia-Feng Tsai, Ph.D. chia-feng.tsai@pnnl.gov

Acknowledgment: Portions of this work was supported by NCI grant U24CA210955, NCI EDRN Interagency Agreements (ACN20007-001), a Laboratory Directed Research and Development award

(Y.Z.) from the Pacific Northwest National Laboratory (PNNL), and an Intramural program (Y.Z.) at the Environmental Molecular Sciences Laboratory (grid.436923.9), a DOE Office of Science User Facility

sponsored by the Office of Biological and Environmental Research. PNNL is a multiprogram national laboratory operated by Battelle for the DOE under contract DE-AC05-76RL01830.

Preliminary resultStrategyMimic single-cell proteomics

NoBoost Boost

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14% 17%

Figure 5. The effects of FAIMS-basediMBR approach on mimic single-cellproteome analysis. (a) Tryptic digests ofthree different acute myeloid leukemia(AML) cells were used to mimic single-cell proteome analysis conditions. (b)After using IMBR, the number ofquantifiable peptides increasedsignificantly especially for NoBoostsamples. Without using boostingsamples, the TMT intensity increased (c),resulting in lower CV (%) (d) .

(a) (b) (c) (d)

Figure 6. The effects on IMBR for single-cell proteomeanalysis. (a) Our recently developed nested nanoPOTS (N2)[4]

technology was integrated with the boosting-free andmultiplexed single-cell proteomics analysis. (b) After usingIMBR, the number of quantifiable peptides increasedsignificantly especially for NoBoost samples. Without usingboosting samples, the TMT intensity increased (c), allowing forrobust separation of different types of cells in the PCA analysis(d).

(a)

(b)

Conclusions

• Isotopic leakage: Without using the boosting material, the quantitation error caused by impurity of the boosting samples can be

minimized, improving the quantitation performance of single-cell proteome analysis.

• Ion sampling: Without using boosting material, the C-trap can allow all the ions from the single cells transmit to orbitrap for scanning,

increasing the TMT intensity of peptides from the single cells.

• Coverage: By using accurate RT, m/z and FAIMS CV as 3-D features for match between run, the coverage of single-cell proteomics

effectively increases without the need for boosting materials.

Raw

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C10

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70 % valid in all samples

NoBoost Boost

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9.59.2 10.39.5

8.8 9.7

98 cells 98 cells

# In the preliminary data for real single-cell proteomics,we’ve only thus far adapted isobaric match betweenrun (iMBR) approach without using FAIMS interface toincrease the proteome coverage without using aboosting sample. The boosting-free single-cellproteome analysis with FAIMS interface is still ongoing.

TMT Cell tye Input (ng)

126 MOLM-14 0.1

127N K562 0.1

127C CMK 0.1

128N MOLM-14 0.1

128C K562 0.1

129N CMK 0.1

129C MOLM-14 0.1

130N K562 0.1

130C CMK 0.1

131N MOLM-14 0.1

131C K562 0.1

132N CMK 0.1

132C MOLM-14 0.1

133N Empty 0.1

133C CMK 0.1

134N Ref/Boost 0.5/10