Picoflow Liquid Chromatography–Mass Spectrometry for Ultrasensitive Bottom-Up Proteomics Using 2-μm-i.d. Open Tubular Columns

Abstract

In many areas of application, key objectives of chemical separation and analysis are to minimize the sample quantity while maximizing the chemical information obtained. Increasing measurement sensitivity is especially critical for proteomics research, especially when processing trace samples and where multiple measurements are desired. A rich collection of technologies has been developed, but the resulting sensitivity remains insufficient for achieving in-depth coverage of proteomic samples as small as single cells. Here, we combine picoliter-scale liquid chromatography (picoLC) with mass spectrometry (MS) to address this issue. The picoLC employs a 2-μm-i.d. open tubular column to reduce the sample input needed to greatly increase the sensitivity achieved using electrospray ionization (ESI) with MS. With this picoLC-MS system, we show that we can identify ∼1000 proteins reliably using only 75 pg of tryptic peptides, representing a 10–100-fold sensitivity improvement compared with the state-of-the-art liquid chromatography (LC) or capillary electrophoresis (CE)-MS methods. PicoLC-MS extends the limit of separation science and is expected to be a powerful tool for single cell proteomics.

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

“…New microfluidic strategies could be developed to minimize droplet evaporation and increase droplet dispensing precision 43 . The proteome coverages could be improved with lower-flow LC systems 13 , 44 , advanced MS instrumentation 23 , 40 , and data analysis strategies 45 .…”

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation

“…As electrospray (ES) is concentration dependent, sensitivity increases with decreasing flow-rate, however, very low flow systems are challenging to operate robustly and are consequently not widely available [28][29][30][31] . We recently described a chromatography system that decouples sample loading and gradient formation from the LC-MS run and operates at a standardized flow-rate of 1 ul/min for high reproducibility 32 .…”

Single-cell Proteomics: Progress and Prospects

“…More recently, Xiang et al employed 2-µm-i.d. open tubular "picoLC" columns for trace proteomic analyses (64). The separations operated at just 790 pL/min and achieved a respective proteome coverage of 78 and 949 protein groups for 7.5 and 75 pg of bacterial lysate tryptic digest, which is far less protein than is found in a typical mammalian cell.…”