Biology subjects

Huffman, R. G.

Publications and source records attributed to Huffman, R. G..

3 recordsLinked to original sources

bioRxiv2021

Increasing the throughput of sensitive proteomics by plexDIA

Current mass-spectrometry methods enable high-throughput proteomics of large sample amounts, but proteomics of low sample amounts remains limited in depth and throughput. To increase the throughput of sensitive proteomics, we developed an experimental and computational framework, plexDIA, for simultaneously multiplexing the analysis of both peptides and samples. Multiplexed analysis with plexDIA increases throughput multiplicatively with the number of labels without reducing proteome coverage or quantitative accuracy. By using 3-plex nonisobaric mass tags, plexDIA enables quantifying 3-fold more protein ratios among nanogram-level samples. Using 1 hour active gradients and first-generation Q Exactive, plexDIA quantified about 8,000 proteins in each sample of labeled 3-plex sets. plexDIA also increases data completeness, reducing missing data over 2-fold across samples. We applied plexDIA to quantify proteome dynamics during the cell division cycle in cells isolated based on their DNA content; plexDIA detected many classical cell cycle proteins and discovered new ones. When applied to single human cells, plexDIA quantified about 1,000 proteins per cell and achieved 98 % data completeness within a plexDIA set while using about 5 min of active chromatography per cell. These results establish a general framework for increasing the throughput of sensitive and quantitative protein analysis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/467007v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@144bba6org.highwire.dtl.DTLVardef@17bbf20org.highwire.dtl.DTLVardef@140f497org.highwire.dtl.DTLVardef@179faa9_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2021

Droplet sample preparation for single-cell proteomics applied to the cell cycle

Many biological processes, such as the cell division cycle, are reflected in protein covariation across single cells. This covariation can be quantified and interpreted by single-cell mass-spectrometry (MS) with sufficiently high throughput and accuracy. Towards this goal, we developed nPOP, a method that uses piezo acoustic dispensing to isolate individual cells in 300 picoliter volumes and performs all subsequent sample preparation steps in small droplets on a fluorocarbon-coated slide. This design enabled simultaneous sample preparation of thousands of single cells, including lysing, digesting, and labeling individual cells in volumes of 8-20 nl. Protein covariation analysis identified cell-cycle dynamics that were similar across cell types and dynamics that differed between cell types, even within sub-populations of melanoma cells defined by markers for drug-resistance priming. The melanoma cells expressing these markers accumulated in the G1 phase of the cell cycle, displayed distinct protein covariation across the cell cycle, accumulated glycogen, and had lower abundance of glycolytic enzymes. The non-primed melanoma cells exhibited gradients of protein abundance and covariation, suggesting transition states. These results were validated by different MS methods. Together, they demonstrate that protein covariation across single cells may reveal functionally concerted biological differences between closely related cell states.

bioengineering↗

bioRxiv2021

Multiplexed single-cell proteomics using SCoPE2

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying over 1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Isobaric carrier based multiplexed single-cell proteomics is a scalable, reliable, and cost-effective method that can be fully automated and implemented on widely available equipment. It uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. Here we describe an automated Single Cell ProtEomics (SCoPE2) workflow that allows analyzing about 200 single cells per 24 hours using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis. SCoPE2 Protocol O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=40 SRC="FIGDIR/small/435034v2_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@b0d69dorg.highwire.dtl.DTLVardef@1da07d4org.highwire.dtl.DTLVardef@1381c5borg.highwire.dtl.DTLVardef@cdb087_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗