Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome Profiling

Abstract

Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report a fully automated platform termed autoPOTS (automated preparation in one pot for trace samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was utilized for one-pot sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30 μm i.d. nanoLC columns. We used autoPOTS to analyze 1–500 HeLa cells and observed only a moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ∼130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.

Ultrasensitive NanoLC-MS of Subnanogram Protein Samples Using Second Generation Micropillar Array LC Technology with Orbitrap Exploris 480 and FAIMS PRO

“…Therefore, the 60 min gradient LC method was used to explore the proteome coverage that could be achieved for sample loads up to 5 ng of HeLa tryptic peptides. 1 , 3 , 13 , 27 …”

“… 30 However, one should be aware of the fact that the numbers obtained in the current technical note represent what could be feasible when all pieces of a single-cell proteomics workflow have been carefully optimized. Even though several innovative breakthroughs have been described that allow near lossless single-cell sample preparation, 1 , 3 , 13 , 14 the analysis of subnanogram aliquots of preprepared bulk samples will inevitably be more reproducible and result in a higher yield. This has been illustrated by the results reported in a recent article by Cong et al, 15 where significant improvements in low input proteome coverage were described by implementing ultralow flow (ULF) LC (20 nL/min) combined with FAIMS and Orbitrap Eclipse MS.…”

Ultra-sensitive nanoLC-MS of sub nanogram protein samples using second generation micro pillar array LC technology with Orbitrap Exploris 480 and FAIMS PRO

“…However, one should be aware of the fact that the numbers obtained in current manuscript represent what could be feasible when all pieces of a single cell proteomics workflow have been carefully optimized. Even though several innovative breakthroughs have been described that allow near loss-less single-cell sample preparation 1,3,13,14 , the analysis of sub nanogram aliquots of pre-prepared bulk samples will inevitably be more reproducible and result in a higher yield. This has been illustrated by the results reported in a recent article by Cong et al 15 , where significant improvements in low input proteome coverage were described by implementing ultra-low flow (ULF) LC (20 nL/min) combined with FAIMS and Orbitrap Eclipse MS.…”

“…Therefore the entire proteomics workflow needs to be optimized carefully to achieve ultrasensitive analysis and near loss-less processing of protein samples [9][10][11] . Recent breakthroughs in the field of low input LC-MS/MS based proteomics have been obtained in parallel by several independent groups, each using a unique approach to tackle the challenges associated with comprehensive single cell proteome analysis 1,3,[12][13][14][15] . The key aspects to the successful implementation seem to be a combination of miniaturized and automated sample preparation methods with ultrasensitive LC-MS/MS analysis performed at very low LC flow rates (≤ 100 nL/min), additional ion mobility separation and the latest generation of hybrid or tribrid MS-MS instruments.…”

Temporal variation in lymphocyte proteomics

“…Samples were analyzed using global proteomics for low input samples. First, protein was extracted using automated digestion and nanoLC-MS/MS as described by Liang et al 17 . Cells were sonicated with 4 μ l of 0.1% DDM (n-dodecyl β -D-maltoside) for 5 minutes and centrifuged at 1500 rpm for 1 minute after sorting.…”