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. 2021 Sep 3;20(9):4452-4461.
doi: 10.1021/acs.jproteome.1c00403. Epub 2021 Aug 5.

Facile One-Pot Nanoproteomics for Label-Free Proteome Profiling of 50-1000 Mammalian Cells

Kendall Martin  1   2 , Tong Zhang  1 , Tai-Tu Lin  1 , Amber N Habowski  3 , Rui Zhao  4 , Chia-Feng Tsai  1 , William B Chrisler  1 , Ryan L Sontag  1 , Daniel J Orton  1 , Yong-Jie Lu  5 , Karin D Rodland  1 , Bin Yang  1   2 , Tao Liu  1 , Richard D Smith  1 , Wei-Jun Qian  1 , Marian L Waterman  3 , H Steven Wiley  4 , Tujin Shi  1
Affiliations

Facile One-Pot Nanoproteomics for Label-Free Proteome Profiling of 50-1000 Mammalian Cells

Kendall Martin et al. J Proteome Res. .

Abstract

Recent advances in sample preparation enable label-free mass spectrometry (MS)-based proteome profiling of small numbers of mammalian cells. However, specific devices are often required to downscale sample processing volume from the standard 50-200 μL to sub-μL for effective nanoproteomics, which greatly impedes the implementation of current nanoproteomics methods by the proteomics research community. Herein, we report a facile one-pot nanoproteomics method termed SOPs-MS (surfactant-assisted one-pot sample processing at the standard volume coupled with MS) for convenient robust proteome profiling of 50-1000 mammalian cells. Building upon our recent development of SOPs-MS for label-free single-cell proteomics at a low μL volume, we have systematically evaluated its processing volume at 10-200 μL using 100 human cells. The processing volume of 50 μL that is in the range of volume for standard proteomics sample preparation has been selected for easy sample handling with a benchtop micropipette. SOPs-MS allows for reliable label-free quantification of ∼1200-2700 protein groups from 50 to 1000 MCF10A cells. When applied to small subpopulations of mouse colon crypt cells, SOPs-MS has revealed protein signatures between distinct subpopulation cells with identification of ∼1500-2500 protein groups for each subpopulation. SOPs-MS may pave the way for routine deep proteome profiling of small numbers of cells and low-input samples.

Keywords: SOPs-MS; colon crypt cells; nanoproteomics; small numbers of cells; surfactant DDM.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Schematic diagram of the SOPs-MS work flow at the standard sample processing volume (∼50 μL). Small subpopulations of cells are isolated by FACS into either PCR tubes or 96-well PCR plates (top panel). Surfactant DDM-assisted one-pot sample processing is conducted in either a single PCR tube or a PCR well without sample transfer including cell lysis, protein denaturation, reduction and alkylation (these two steps are optional), and trypsin digestion (middle panel). Prior to LC–MS analysis, the sample volume is reduced to ∼20 μL for full injection. The cap of the PCR tube is removed, and the tube is inserted into the LC vial to avoid transfer loss, and the 96-well cap matt is used to cover the 96-well plate for automatic injection without sample transfer (bottom panel). Samples are analyzed by standard LC–MS platforms for quantitative label-free proteomic analysis.
Figure 2.
Figure 2.
Evaluation of SOP performance at different processing volumes. (A) Number of identified unique peptides with MaxQuant. (B) Number of identified protein groups with MaxQuant. (C) Violin plots showing the distribution of the CVs of LFQ intensities at the peptide level. (D) Violin plots showing the distribution of CVs of LFQ intensities at the protein level. Red bar: MS/MS spectra only; blue bar: the combined MS/MS and MBR. Data are shown as the mean value ± SD. In (C,D), red horizontal lines indicate the median CVs of 14.7% for the peptides and 9.9% for the protein groups across different processing volumes.
Figure 3.
Figure 3.
Evaluation of SOPs-MS using FACS-sorted MCF10A cells. (A) Number of identified unique peptides for 50–1000 cells. (B) Number of identified protein groups for 50–1000 cells. (C) Venn diagram showing the number of protein groups identified from each of 3 biological replicates for 50 cells. (D) Pairwise correlation of protein LFQ intensities between any two replicates with the Pearson correlation coefficient. Red bar: MS/MS spectra only; blue bar: combined MS/MS and MBR. Data are shown as the mean value ± SD.
Figure 4.
Figure 4.
Application of SOPs-MS for analysis of small subpopulations of colon crypt cells. (A) Number of identified protein groups for the five small subpopulation cells. Red bar: MS/MS spectra only; blue bar: combined MS/MS and MBR. Data are shown as the mean value ± SD. Below is the average cell number for each subpopulation. (B) Violin plots of the distribution of CVs for proteomic analysis of each of the subpopulation cells. (C) Heatmap with unsupervised clustering to show relative abundance for each protein group identified by the MaxQuant MBR for all the five small subpopulations with three biological replicates per subpopulation.
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