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runsheng_zheng
Team TFS
Team TFS

The state-of-the-art low-flow Thermo ScientificTM Vanquish™ Neo liquid chromatography (LC) system, coupled with electrospray ionization-based Thermo ScientificTM Orbitrap™ Exploris 480 mass spectrometry (MS) technologies, can provide the sensitivity and throughput necessary to support the growing need for analyzing low- and limited-sample amounts, such as those from single-cell. In particular, the capacity to generate precise low-flow gradient separations at high pressure for low internal diameter separation columns, leak-free connections, and optimal data acquisition strategy are essential for consistent data quality with sufficient sample analysis throughput.

 

LCMS Sensitivty Poster Social.jpgSeveral aspects of limited sample amounts analysis play a significant role in getting acceptable sensitivity and throughput. First, the flow rate should be optimized to find the balance between the sensitivity and the throughput. Second, the LC setup must allow analysis in a pain-free manner without wasting MS time. Third, MS acquisition parameters should be optimized to accommodate low signal intensity from small sample amounts. Considering all the factors, we developed 5 nLC-MS methods with a PepMap C18 50 µm I.D. x 15 cm column operated at 1,500 bar for sample loading and 100 nL/min for gradient separation, to demonstrate the high-sensitivity and high-throughput (24-72 samples/day) performance of the Vanquish Neo UHPLC system coupled to an Orbitrap Exploris 480 mass spectrometer with a FAIMS Pro interface.

 

We confidently identified ~1,500 protein groups from 250 pg diluted HeLa standard with a 30-minute gradient in data-dependent acquisition (DDA) using the SEQUEST + INFERYS rescoring node (without match-between-runs (MBR)), which, to the author’s knowledge, represents the most comprehensive DDA data to date1. More excitingly, by identifying ~800 protein groups in a 10-min gradient (72 samples/day) we have shown its great potential for higher performance analysis in DDA and data-independent acquisition (DIA) using AI-driven database search algorithms. Interestingly but not surprisingly, 10 percent - 20 percent more peptide and protein identifications were gained from a method with a faster scan speed by using half of the original MS2 injection time. However, an acquisition with a quarter of the original injection time impacted the performance reversely, where more MS2 did not translate into peptide spectral match (PSM) and, therefore, peptide and protein identifications. It clearly illustrates that sufficient time for ion accumulation with high MS resolution, not the scan speed, is more crucial for analyzing precursor fragments and spectrum identification for low sample amounts analysis.

 

By employing an advanced AI-driven algorithm to deconvolute the spectra, we boosted the protein group identification by 120 percent with a wide window acquisition strategy (10 – 12 Da isolation window), resulting in 1,800 protein group identifications from 250 pg HeLa digest in a 10-minute gradient. A further experiment with the DIA strategy (40 Da isolation window) resulted in >2,100 protein groups identification on average with Spectronaut 16, where DIA-NN 18 outperformed with ca. 25 percent more IDs (ca. 2,700 IDs).

Overall, we evaluated the suitability of the Vanquish Neo UHPLC system and Orbitrap Exploris 480 MS for the analysis of limited sample amounts, which affords 24 -72 samples/day throughput with high MS utilization and industry-leading protein coverage in DDA and DIA modes.

 

For more details, read the poster: A high-sensitivity high-throughput LCMS platform for single-cell proteomics and low sample amount an...

 

1Karel Stejskal, Jeff Op de Beeck, etc. Ultrasensitive NanoLC-MS of Subnanogram Protein Samples Using Second Generation Micropillar Array LC Technology with Orbitrap Exploris 480 and FAIMS PRO Anal. Chem. 2021, 93, 25, 8704–8710

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Last update:
‎11-29-2022 10:32 AM
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