Optimizing single cell proteomics using trapped ion mobility spectrometry for label-free experiments

Mun, Dong-Gi; Bhat, Firdous Ahmad; Ding, Husheng; Madden, Benjamin J.; Natesampillai, Sekar; Badley, Andrew D.; Johnson, Kenneth L.; Pandey, Akhilesh

doi:10.1039/d3an00080j

Cited by 7 publications

(5 citation statements)

References 42 publications

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“…To gain molecular insights into SGSPC, we performed proteome profiling of single cells using trapped ion mobility spectrometry (TIMS) in conjunction with a time-of-flight mass spectrometer operated in parallel accumulation-serial fragmentation (PASEF) mode 40,41 . This cutting-edge methodology enhances proteome coverage from low-input samples, enabling the generation of label-free, unbiased liquid Chromatography with tandem mass spectrometry (LC-MS/MS)-based single-cell proteomic profiles of SGSPC-enriched single cells (Figure 5A) 42,43 . To accomplish this, we used our strategy to enrich for progenitors FACS sorting viable (DAPI-) CD45/CD31-EpCAM+ progenitor-enriched cells (referred hereafter as progenitor-enriched single cells) through stringent gating procedures.…”

Section: Unbiased Mass Spectrometry-based Single-cell Proteomic Profi...mentioning

confidence: 99%

“…Following FACS-based separation of viable progenitor-rich salivary single cells from SMG and PG, cells were sorted, and reactions were performed on a 384-well plate using the cellenONE system (Cellenion, France) 42 . Lysis buffer was prepared with the concentration of 0.2% DDM (324355-1GM, Millipore, USA), 100 mM TEAB (T7408-500ML, Sigma-Aldrich, USA) and 2 ng/µl trypsin protease (90057, Thermo Scientific, USA).…”

Section: Sample Preparation For Salivary Single-cell Proteomicsmentioning

confidence: 99%

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The Mayo Clinic Salivary Tissue-Organoid Biobanking: A Resource for Salivary Regeneration Research

Aalam,

Varela,

Khaderi

et al. 2024

Preprint

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The salivary gland (SG) is an essential organ that secretes saliva, which supports versatile oral function throughout life, and is maintained by elusive epithelial stem and progenitor cells (SGSPC). Unfortunately, aging, drugs, autoimmune disorders, and cancer treatments can lead to salivary dysfunction and associated health consequences. Despite many ongoing therapeutic efforts to mediate those conditions, investigating human SGSPC is challenging due to lack of standardized tissue collection, limited tissue access, and inadequate purification methods. Herein, we established a diverse and clinically annotated salivary regenerative biobanking at the Mayo Clinic, optimizing viable salivary cell isolation and clonal assays in both 2D and 3D-matrigel growth environments. Our analysis identified ductal epithelial cells in vitro enriched with SGSPC expressing the CD24/EpCAM/CD49f+ and PSMA- phenotype. We identified PSMA expression as a reliable SGSPC differentiation marker. Moreover, we identified progenitor cell types with shared phenotypes exhibiting three distinct clonal patterns of salivary differentiation in a 2D environment. Leveraging innovative label-free unbiased LC-MS/MS-based single-cell proteomics, we identified 819 proteins across 71 single cell proteome datasets from purified progenitor-enriched parotid gland (PG) and sub-mandibular gland (SMG) cultures. We identified distinctive co-expression of proteins, such as KRT1/5/13/14/15/17/23/76 and 79, exclusively observed in rare, scattered salivary ductal basal cells, indicating the potential de novo source of SGSPC. We also identified an entire class of peroxiredoxin peroxidases, enriched in PG than SMG, and attendant H2O2-dependent cell proliferation in vitro suggesting a potential role for PRDX-dependent floodgate oxidative signaling in salivary homeostasis. The distinctive clinical resources and research insights presented here offer a foundation for exploring personalized regenerative medicine.

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Section: Unbiased Mass Spectrometry-based Single-cell Proteomic Profi...mentioning

confidence: 99%

Section: Sample Preparation For Salivary Single-cell Proteomicsmentioning

confidence: 99%

The Mayo Clinic Salivary Tissue-Organoid Biobanking: A Resource for Salivary Regeneration Research

Aalam,

Varela,

Khaderi

et al. 2024

Preprint

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“…When loading 1 ng of HeLa peptides onto the timsTOF SCP instrument, Mann’s group identified over 550 proteins in the DDA method and over 3000 proteins in the DIA method combined with low-flow-rate chromatography . Pandey’s group further optimized the parameters of the DDA-PASEF method and was able to detect 1116 proteins from 10 sorted human primary T cells . The timsTOF SCP instrument serves as an excellent platform for proteomics research involving limited sample material.…”

Section: Introductionmentioning

confidence: 99%

“…37 Pandey's group further optimized the parameters of the DDA-PASEF method and was able to detect 1116 proteins from 10 sorted human primary T cells. 38 The timsTOF SCP instrument serves as an excellent platform for proteomics research involving limited sample material. However, to the best of our knowledge, there has been no thorough parameter optimization study for the diaPASEF method, and only a limited number of studies have focused on enhancing the sensitivity of the timsTOF SCP instrument and evaluating protein quantification for data acquired in the diaPASEF method.…”

Section: ■ Introductionmentioning

confidence: 99%

Evaluation of Protein Identification and Quantification by the diaPASEF Method on timsTOF SCP

Wang,

Tan,

et al. 2024

J. Am. Soc. Mass Spectrom.

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Accurate and precise quantification is crucial in modern proteomics, particularly in the context of exploring low-amount samples. While the innovative 4D-data-independent acquisition (DIA) quantitative proteomics facilitated by timsTOF mass spectrometers gives enhanced sensitivity and selectivity for protein identification, the diaPASEF (parallel accumulation-serial fragmentation combined with data-independent acquisition) parameters have not been systematically optimized, and a comprehensive evaluation of the quantification is currently lacking. In this study, we conducted a thorough optimization of key parameters on a timsTOF SCP instrument, including sample loading amount (50 ng), ramp/accumulation time (140 ms), isolation window width (20 m/z), and gradient time (60 min). To further improve the identification of proteins in low-amount samples, we utilized different column settings and introduced 0.02% n-dodecyl-β-D-maltoside (DDM) in the sample reconstitution solution, resulting in a remarkable 19fold increase in protein identification at the single-cell-equivalent level. Moreover, a comprehensive comparison of protein quantification using a tandem mass tag reporter (TMT-reporter), complement TMT ions (TMTc), and diaPASEF revealed a strong correlation between these methods. Both diaPASEF and TMTc have effectively addressed the issue of ratio compression, highlighting the diaPASEF method's effectiveness in achieving accurate quantification data compared to TMT reporter quantification. Additionally, an in-depth analysis of in-group variation positioned diaPASEF between the TMT-reporter and TMTc methods. Therefore, diaPASEF quantification on the timsTOF SCP instrument emerges as a precise and accurate methodology for quantitative proteomics, especially for samples with small amounts.

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“…When a few scientists were entertaining the idea of proteomic measurements of single individual cells, the vast majority of the field deemed this endeavor impossible. Today, we know that indeed it is very much possible albeit still challenging to do single-cell proteomics (SCP) [14][15][16][17][18][19][20][21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Immunopeptidomics in the Era of Single-Cell Proteomics

Mayer,

Mechtler

2023

Biology

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Immunopeptidomics, as the analysis of antigen peptides being presented to the immune system via major histocompatibility complexes (MHC), is being seen as an imperative tool for identifying epitopes for vaccine development to treat cancer and viral and bacterial infections as well as parasites. The field has made tremendous strides over the last 25 years but currently still faces challenges in sensitivity and throughput for widespread applications in personalized medicine and large vaccine development studies. Cutting-edge technological advancements in sample preparation, liquid chromatography as well as mass spectrometry, and data analysis, however, are currently transforming the field. This perspective showcases how the advent of single-cell proteomics has accelerated this transformation of immunopeptidomics in recent years and will pave the way for even more sensitive and higher-throughput immunopeptidomics analyses.

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Optimizing single cell proteomics using trapped ion mobility spectrometry for label-free experiments

Abstract: We present optimized settings for ramp times and ion mobility range in trapped ion mobility spectrometry experiments involving single cell analysis.

Cited by 7 publications

References 42 publications

The Mayo Clinic Salivary Tissue-Organoid Biobanking: A Resource for Salivary Regeneration Research

The Mayo Clinic Salivary Tissue-Organoid Biobanking: A Resource for Salivary Regeneration Research

Evaluation of Protein Identification and Quantification by the diaPASEF Method on timsTOF SCP

Immunopeptidomics in the Era of Single-Cell Proteomics

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