Parallelization with Dual-Trap Single-Column Configuration Maximizes Throughput of Proteomic Analysis

Abstract

Proteomic analysis on the scale that captures population and biological heterogeneity over hundreds to thousands of samples requires rapid mass spectrometry methods, which maximize instrument utilization (IU) and proteome coverage while maintaining precise and reproducible quantification. To achieve this, a short liquid chromatography gradient paired to rapid mass spectrometry data acquisition can be used to reproducibly quantify a moderate set of analytes. Highthroughput profiling at a limited depth is becoming an increasingly utilized strategy for tackling large sample sets but the time spent on loading the sample, flushing the column(s), and re-equilibrating the system reduces the ratio of meaningful data acquired to total operation time and IU. The dual-trap single-column configuration (DTSC) presented here maximizes IU in rapid analysis (15 min per sample) of blood and cell lysates by parallelizing trap column cleaning and sample loading and desalting with the analysis of the previous sample. We achieved 90% IU in low microflow (9.5 μL/min) analysis of blood while reproducibly quantifying 300−400 proteins and over 6000 precursor ions. The same IU was achieved for cell lysates and over 4000 proteins (3000 at CV below 20%) and 40,000 precursor ions were quantified at a rate of 15 min/sample. Thus, DTSC enables high-throughput epidemiological blood-based biomarker cohort studies and cell-based perturbation screening.

High-Throughput Single-Cell Proteomic Analysis of Organ-Derived Heterogeneous Cell Populations by Nanoflow Dual-Trap Single-Column Liquid Chromatography

“…Operation of two analytical columns in parallel, where a sample is loaded on one column while a second sample is analyzed, elegantly increases IU but requires a specialized ion source that accommodates two emitters and an LC with two analytical pumps . Our dual-trap single-column (DTSC) platform optimized for the nano flow rate (500 nL/min, nanoDTSC) increases the throughput of single-cell analysis by parallelizing sensitive high ion accumulation time (166 ms) DIA-PASEF data acquisition on the timsTOF-SCP (Bruker) with resuspension, loading, and desalting of the subsequent sample . NanoDTSC does not require specialized instrumentation, so it is widely accessible; here, an Ultimate 3000 nanoRSLC (Thermo) and the standard Bruker captive spray ionization source were used.…”

High Throughput Single Cell Proteomic Analysis of Organ Derived Heterogeneous Cell Populations by Nanoflow Dual Trap Single Column Liquid Chromatography

“…The dual trap single column configuration (DTSC) 11 was adapted for single cell analysis ( Fig. S1 ) using cartridge trapping columns with a 0.17 μL media bed (EXP2 from Optimize Technologies) packed with 10 μm diameter, 100 Å pore PLRP-S (Agilent) beads, and a PepSep 15 cm × 75 μm analytical column packed with 1.9 μm C18 solid phase (Bruker).…”

“…9 Our dual trap single column (DTSC) platform optimized for the nano flowrate (500 nL/min, nanoDTSC) increases the throughput of single cell analysis by parallelizing sensitive high ion accumulation time (166 ms) DIA-PASEF data acquisition on the timsTOF-SCP (Bruker) with resuspension, loading, and desalting of the subsequent sample. 11 NanoDTSC does not require specialized instrumentation making it widely accessible; here an Ultimate 3000 nanoRSLC (Thermo) with the standard Bruker captive spray ionization source were used. Experimental Details Liquid Chromatography: The dual trap single column configuration (DTSC) 11 was adapted for single cell analysis (Fig.…”

“…11 NanoDTSC does not require specialized instrumentation making it widely accessible; here an Ultimate 3000 nanoRSLC (Thermo) with the standard Bruker captive spray ionization source were used. Experimental Details Liquid Chromatography: The dual trap single column configuration (DTSC) 11 was adapted for single cell analysis (Fig. S1) using cartridge trapping columns with a 0.17 µL media bed (EXP2 from Optimize Technologies) packed with 10 µm diameter, 100 Å pore PLRP-S (Agilent) beads, and a PepSep 15 cm x 75 µm analytical column packed with 1.9 µm C18 solid phase (Bruker).…”

Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics

“…Dual trap single column (DTSC) 17 was adapted for nanoflow to enable single cell analysis 18 . The trapping columns were 0.17 µL media bed (EXP2 from Optimize Technologies) packed with 10 µm diameter, 100 Å pore PLRP-S (Agilent) beads, and the analytical column was a PepSep 15 cm x 75 µm packed with 1.9 µm C18 stationary phase (Bruker).…”