Automated container-less cell processing method for single-cell proteomics

Abstract

Single-cell genomics and transcriptomics studies enabled us to characterize cell heterogeneity in various tissues, which helped us to better understand the biological system and disease progression. Single-cell proteomics, which directly measures the protein expression level, has the potential to further enhance our knowledge by providing not only a more direct measurement but also crucial information cannot be captured by genomics or transcriptomics study, such as protein activation states and post-translation modification events. One of the main challenges of single-cell proteomics is the large sample loss during sample preparation, which is largely unavoidable using standard proteomics protocols. Protein and peptide loss to the container surface is a well-known phenomenon but often overlooked in larger-scale (>1 microgram) proteomics experiments. When it comes to single-cell proteomics with only picograms of protein samples, this loss becomes non-negligible and often dictates the outcomes of the experiment. More importantly, sample processing through multiple pipette tips and containers often introduces random errors, which undermine the ability to detect true heterogenous cellular events. To solve these problems and further improve the throughput and reproducibility of the single-cell proteomics experiments, we developed an automated container-less cell processing platform, utilizing acoustic levitation to process cell samples in the air. Our platform automatically performs cell lysis, protein reduction, alkylation, digestion, and peptide labeling in the air, without any sample transfer step or container. The digested and labeled peptides are then directly injected into the capillary LC-MS/MS system for analysis, eliminating manual steps and conserving most of the sample materials for proteomics analysis. Our initial test shows at least 30% improvement in peptide signals over conventional methods. This process can be performed in parallel to further improve sample processing throughput.

Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags

“…While the prospect of SCP was once a distant goal, advances in instrumentation and data acquisition strategies have made SCP a reality. ,,− Developments in sample processing that minimize protein loss have been crucial to maximize the amount of peptide ions entering the mass spectrometer. ,,− Isobaric labeling approaches with tandem mass tags (TMTs) have become a centerpiece of SCP through methods such as SCoPE-MS, which use carrier channels to boost MS 1 -level detection and reporter ions in MS/MS scans to provide relative quantitation of labeled proteins of individual cells. , TMT enables sample multiplexing and quantification of proteomes across experimental conditions. − …”

“…In single-cell proteomics, the limited starting material produces low ion counts and challenges in detecting low-abundance peptides. , Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of peptide ions that make it from ion source to the detector. , Herein, we report infrared-TMT (IR-TMT), an MS 3 -based quantitative proteomics method employing infrared multiphoton photoactivation and reporter ion parking to maximize sensitivity and accuracy of isobaric tagging studies. As an extension of our previous work, we applied IR photoactivation and an RF ion parking waveform to TMTpro labeled peptides and applied the method to single-cell proteomics to greatly boost the generation of quantitative reporter ions.…”

HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics

“…Liquid chromatography–mass spectrometry (LC–MS) is increasingly utilized for unbiased analysis of the proteome of individual cells . The increased speed and sensitivity of modern mass spectrometers, , along with improved sample preparation, − separations, − and experimental design − have allowed for deeper analysis in single-cell proteomics (SCP). SCP can be broadly categorized as advancing along two tracks: label-free and label-based workflows.…”

Simple Tool for Rapidly Assessing the Quality of Multiplexed Single Cell Proteomics Data

“…2−5 Although other single cell technologies such as single cell RNaseq have been sorting and analyzing single cells for over a decade with increasingly sophisticated technology, relatively little of this can be applied to proteomics approaches. 6,7 New technologies such as nanoPOTs, 8 nPOP, 4 LevCell 9 and ProteoChip 5 are improving reproducible peptide recovery from cells, however technical challenges in sample preparation may be the biggest limiter to entry into single cell proteomics today. Single human cells and the volumes of reagents used in their preparation and analysis can be affected by factors as ubiquitous as relative humidity and static electricity.…”

“…Data dependent LCMS analysis of these multiplexed samples leads to the fragmentation and identification of peptides from the carrier channel. The isobaric reporter ion fragments corresponding to labels applied to single cells, if within the intrascan linear dynamic range of the instrument, can be used for assigning both peptide identification and quantification to the cells themselves. − Although other single cell technologies such as single cell RNaseq have been sorting and analyzing single cells for over a decade with increasingly sophisticated technology, relatively little of this can be applied to proteomics approaches. , New technologies such as nanoPOTs, nPOP, LevCell and ProteoChip are improving reproducible peptide recovery from cells, however technical challenges in sample preparation may be the biggest limiter to entry into single cell proteomics today. Single human cells and the volumes of reagents used in their preparation and analysis can be affected by factors as ubiquitous as relative humidity and static electricity .…”