Abstract
To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system - termed Evosep One - analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage.
Highlights
State-of-the-art workflows begin with a robust sample preparation to digest proteins and harvest purified peptides [2], which are separated by a liquid chromatography (LC)1 system before they are analyzed by a mass spectrometer (MS)
Note that an entire gradient can be stored in a several meters long fused silica capillary - already containing the individual peptides at the organic content where they elute from the C18 material
For the top 70% of the proteome by abundance, data completeness was close to 100%. These results indicate that the short gradients enabled by the Evosep One can very efficiently be combined with Data independent analysis (DIA) for high-throughput and in-depth acquisition of proteomic data
Summary
To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump This simplified design enables robust operation over thousands of sample injections. In the quest for ever increasing chromatographic separation power, columns have become longer and particle sizes smaller - reaching the sub 2 m range This may require pump pressures more than 1000 bar, presenting great engineering challenges for both the pumps and the entire LC system, often limiting robustness in routine operation. Irreproducibility of retention times within and between laboratories severely limits strategies that rely on the transfer of accurate retention times, especially targeted proteomics [10], data independent acquisition [11] and “match between runs” at the MS level [12, 13]
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