A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust ProteomicsTo 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. 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. Bottom-up proteomics is a highly successful and generic technology, which now allows the analysis of complex samples ranging from bacteria through cell line systems and even human tissue samples (1Aebersold R. Mann M. Mass-spectrometric exploration of proteome structure and function.Nature. 2016; 537: 347-355Crossref PubMed Scopus (1105) Google Scholar). State-of-the-art workflows begin with a robust sample preparation to digest proteins and harvest purified peptides (2Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (991) Google Scholar), which are separated by a liquid chromatography (LC) 1The abbreviation used is:LCliquid chromatography. 1The abbreviation used is:LCliquid chromatography. system before they are analyzed by a mass spectrometer (MS). Established software solutions automatically interpret the acquired spectra, generating lists of thousands of quantified proteins (3Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar, 4Bekker-Jensen D.B. Kelstrup C.D. Batth T.S. Larsen S.C. Haldrup C. Bramsen J.B. Sørensen K.D. Høyer S. Ørntoft T.F. Andersen C.L. Nielsen M.L. Olsen J.V. An optimized shotgun strategy for the rapid generation of comprehensive human proteomes.Cell Systems. 2017; 4: 587-599Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 5Kelstrup C.D. Bekker-Jensen D.B. Arrey T.N. Hogrebe A. Harder A. Olsen J.V. Performance evaluation of the Q Exactive HF-X for shotgun proteomics.J Proteome Res,. 2018; 17: 727-738Crossref PubMed Scopus (159) Google Scholar, 6Kulak N.A. Geyer P.E. Mann M. Loss-less nano-fractionator for high sensitivity, high coverage proteomics.Mol. Cell. Proteomics. 2017; (Manuscript in Press, 2017)Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 7Bruderer R. Bernhardt OM Gandhi T Xuan Y Sondermann J Schmidt M Gomez-Varela D Reiter L Optimization of experimental parameters in data-independent mass spectrometry significantly increases depth and reproducibility of results.Mol. Cell. Proteomics. 2017; 16: 2296-2309Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 8Meier F. Geyer P.E. Virreira Winter S. Cox J. Mann M. BoxCar acquisition method enables single-shot proteomics at a depth of 10,000 proteins in 100 minutes.Nat. Methods. 2018; 10.1038/s41592-018-0003-5Crossref Scopus (218) Google Scholar). liquid chromatography. liquid chromatography. The current performance level is a result of improvements not only in the mass spectrometric components but also the chromatographic part of the LC-MS workflow. In the quest for ever increasing chromatographic separation power, columns have become longer and particle sizes smaller - now reaching the sub 2 μm 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. Thus, chromatography remains a weak link in MS-based proteomics workflows, leading to calls for new approaches (9Riley N.M. Hebert A.S. Coon J.J. Proteomics Moves into the Fast Lane.Cell Syst,. 2016; 2: 142-143Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Furthermore, irreproducibility of retention times within and between laboratories severely limits strategies that rely on the transfer of accurate retention times, especially targeted proteomics (10Picotti P. Aebersold R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions.Nat. Methods. 2012; 9: 555-566Crossref PubMed Scopus (991) Google Scholar), data independent acquisition (11Gillet L.C. Leitner A. Aebersold R. Mass spectrometry applied to bottom-up proteomics: entering the high-throughput era for hypothesis testing.Annu. Rev. Anal. Chem. 2016; 9: 449-472Crossref PubMed Scopus (207) Google Scholar) and “match between runs” at the MS level (12Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell. Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (2688) Google Scholar, 13Geiger T. Wehner A Schaab C Cox J Mann M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins.Mol. Cell. Proteomics. 2012; 11M111.014050Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). There is great interest in applying the increasing power of MS-based proteomics to diagnostic and clinical questions (14Geyer P.E. Holdt L.M. Teupser D. Mann M. Revisiting biomarker discovery by plasma proteomics.Mol Syst Biol,. 2017; 13: 942Crossref PubMed Scopus (390) Google Scholar). “Clinical proteomics”, however, requires far more stability and reproducibility than that available even in the most advanced MS-based proteomics laboratories. Note that irreproducibility and robustness issues are not features of LC-MS per se, as the measurement of small molecules is firmly established in clinical laboratories around the world, which routinely of samples per day. The of these LC systems to the applied in proteomics are column and to and the to robustness in the of proteomics R. Aebersold R. Mass spectrometric protein for biomarker discovery and clinical Rev. 13: PubMed Scopus Google Scholar). the in is and reducing sensitivity at rates, which limits these approaches to a few from high throughput is the for MS-based is to routine clinical current proteomics workflows measurement long gradient In a plasma proteomics in more than a of the time to the system than the the column and steps between the of P.E. N.A. Pichler G. Holdt L.M. Teupser D. Mann M. proteome to human and 2016; 2: Full Text Full Text PDF PubMed Scopus Google Scholar, P.E. S. N. J. S. J.J. Mann M. Proteomics reveals the of on the human plasma 2016; PubMed Scopus Google Scholar). of the current a sample and for liquid for high sample throughput for clinical G. Larsen N. liquid chromatography to for and quantification of protein and Proteome 2014; 13: PubMed Scopus Google Scholar). The of the that are in proteomics for of peptides and J. Mann M. and with for and in Proteome PubMed Scopus Google Scholar, J. Mann M. and for and sample in Chem. PubMed Scopus Google Scholar, Mann M. for analysis of and clinical samples to a depth of PubMed Scopus Google Scholar). of into the of the system, a pump a gradient through the and the The system samples in only as as more than proteins from a HeLa cell in than G. Larsen N. liquid chromatography to for and quantification of protein and Proteome 2014; 13: PubMed Scopus Google Scholar). In with as on to of the in N.A. F. P. N. S. of proteomes and an Proteome PubMed Scopus Google Scholar). for protein the from and of only analytical columns chromatographic separation power of this In the we to the of the while also the features of this by through the to a workflow. In the Evosep One peptides are at and of of from a - termed the gradient with the are in a long loop. 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Thus, the storage the between the and high-pressure The steps are in the and in the the of an LC-MS the of a new LC-MS the of the Evosep One an disposable trap column with and with the at the In the pumps A and a gradient at the that through the disposable trap column, the of interest The of this gradient is to than to that only peptides of interest are the while as and highly to the disposable with from the Furthermore, the of this gradient is to few to and of the more This concept further in The C and D the at the to an to the gradient This the of the that the are on the analytical The gradient with the is into the storage before with the high-pressure pump. In to the the high-pressure pump is and the analytical column is the Evosep One the storage with the high-pressure pump and the gradient with the is the analytical column for high performance separation In to the LC-MS the Evosep One is for the sample by the disposable trap column, the and the the pumps and the of the pumps The to an LC and high in of the system, of the of an in the also in through the software the Evosep One before a sample HeLa were in high with and were an cell and stored at were in by at an of at a of in used to a that are to the of of human protein on for of human protein sample preparation we used the for proteomic samples (2Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (991) Google Scholar), with HeLa to the To and we and analyzed over times peptides of HeLa in this we used the of the Evosep One to a issues were and the system optimized the and in the the only for the of and software The HeLa samples were analyzed on a single column to in the system and the of the by a available and and for at to harvest from a with of the of the The plasma into a and with an sample preparation for Proteome as P.E. N.A. Pichler G. Holdt L.M. Teupser D. Mann M. proteome to human and 2016; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). the as the and the peptides were analyzed with the 200 method with a 2 on a column μm particle for deep proteome analysis were fractionated a μm column on an high-pressure liquid chromatography system at A and were were separated by a gradient from to in by a to in min. 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R. an for and targeted proteomics 26: PubMed Scopus Google Scholar). were from and for a were with 100 steps of in in by times in HeLa peptides were in in The optimized to for the to LC-MS of a Evosep One to an for the more than HeLa and the Evosep One to an Q Exactive HF-X for peptides were separated on the columns with μm and in the data were acquired with a data shotgun method and with a method for the Q Exactive HF-X the Q Exactive HF-X the for the MS in the with a time of and a of at of performed by with a of J.V. for peptide Methods. 4: PubMed Scopus Google Scholar). were performed at a of at 200 with an of and a time of to to of HeLa were at by with and 100 to the The cell by the and for an by for 2 with of on and at by and the with in an ratio of for by with to 2 and further with by with to a of and the peptide on with 2 of by 2 of The were and by and the peptide by at on a peptides were to MS analysis to the samples analyzed on the Evosep an μm column with μm while samples analyzed on the were separated in an μm column with the as The column at an column and with the mass MS were analyzed by the MaxQuant software (3Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar) and lists were the human Proteome without with protein by the J. N. A. Olsen J.V. Mann M. a peptide into the MaxQuant Proteome Res,. PubMed Scopus Google Scholar) with as a and and as The for the 200 method analyzed with the from the of the discovery rate to at the peptide and protein and a of for as to and as and as and a of An mass to and a mass of were independent analysis were with A from the MaxQuant analysis of the analysis of the HeLa and were analyzed peptides and proteins by the software are available in and were with the software S. T. P. A. Hein M. T. Mann M. Cox J. The for comprehensive analysis of 2016; 13: PubMed Scopus Google Scholar) of the MaxQuant in as robust as high-flow LC to gradient from the high high-pressure separation on an analytical in established peptide the peptides are on of J. Mann M. and for and sample in Chem. PubMed Scopus Google of the peptides from the to the and in we elute from the into the loop. This is at pressures of only a few by pumps A and at of to Note that an entire gradient stored in a long - the peptides at the they elute from the a long of 100 μm a of for a analytical column separation of at at the gradient over time in the storage to liquid chromatography system for liquid Mass PubMed Scopus Google Scholar). the high ratio of column with in the this to in a storage in the system we not R. S. Cox J. D. Mann M. A chromatographic method allows of the Cell. Proteomics. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). 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Full Text Full Text PDF PubMed Scopus Google Scholar). established the of we a that far as we leading as the and components were for throughput and robustness requirements in but for software we used the an and used with a to integrate with the MS To operation and we of a HeLa cell digest over times in a issues over time and the only to software In of the within the samples from sample to an of the and the In a we optimized the which in an of the between the and the of the as these issues were From issues to a new column to the concept in in injections. these samples the current the of the A few LC-MS were but this to to of the also the for the only a in that the column of and as further of the the of and were highly and in separation performance of the of were The Evosep One and for high throughput with a on clinical plasma is the most analyzed clinical with of samples is to plasma by of the of To demonstrate clinical of the system, we sample preparation - termed Proteome samples were and on the in a a The measurement time for the samples on the Evosep One than 2 to a throughput of samples per day. over sample and injections of the plasma of over clinical on the of is to from analysis to the we performed a with injections of plasma and The as as and of this to peptides on the and the from the robustness on the we the a of and column to ranging from high throughput of through comprehensive proteomics to the in depth single of complex The by the Evosep system used for complex samples and the longer for more complex Note that the design in the Evosep One also at in the current In common with in proteomics”, we to robustness and throughput over The of and columns with a of not sensitivity this by the sample through the in an optimized for to longer to the chromatographic performance of method a peptide into the complex of a HeLa reaction only the peptides a of proteomic From this data we and retention time for the peptides these data in for the optimized and column for the and sample to demonstrate the throughput on the and used the gradient with the 2 on the column In a single this in 200 data with protein coverage The proteins but of were this is this is not an of range. Furthermore, the proteins were quantified in of that the proteins were from the The high throughput for samples for single protein identification in for for analysis in protein expression in In also for more complex as from the of the system for samples in high we the rapid of high proteomics A is common in the analysis of cell line tissue but with the of a in time as the of on a strategy that high peptide in a without of the and D.B. Kelstrup C.D. Batth T.S. Larsen S.C. Haldrup C. Bramsen J.B. Sørensen K.D. Høyer S. Ørntoft T.F. Andersen C.L. Nielsen M.L. Olsen J.V. An optimized shotgun strategy for the rapid generation of comprehensive human proteomes.Cell Systems. 2017; 4: 587-599Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). to were analyzed in of allowing for high peptide and high and of the high acquisition of mass C.D. Batth T.S. Arrey T.N. A. M. Olsen J.V. and deep proteomes by on a mass Proteome 2014; 13: PubMed Scopus Google Scholar). This in a deep coverage of cell line and tissue on with D.B. Kelstrup C.D. Batth T.S. Larsen S.C. Haldrup C. Bramsen J.B. Sørensen K.D. Høyer S. Ørntoft T.F. Andersen C.L. Nielsen M.L. Olsen J.V. An optimized shotgun strategy for the rapid generation of comprehensive human proteomes.Cell Systems. 2017; 4: 587-599Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). 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D. D. R. S. P. M. G. in by of the 2014; PubMed Scopus Google Scholar) proteomics S. Cox J. and of protein 2016; PubMed Scopus Google Scholar), and for strategies to rapidly and The far used data acquisition data independent acquisition is and R. Bernhardt OM Gandhi T Xuan Y Sondermann J Schmidt M Gomez-Varela D Reiter L Optimization of experimental parameters in data-independent mass spectrometry significantly increases depth and reproducibility of results.Mol. Cell. Proteomics. 2017; 16: 2296-2309Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). In we have to with on and high C.D. Bekker-Jensen D.B. Arrey T.N. Hogrebe A. Harder A. Olsen J.V. Performance evaluation of the Q Exactive HF-X for shotgun proteomics.J Proteome Res,. 2018; 17: 727-738Crossref PubMed Scopus (159) Google Scholar). The Evosep One with between to a to this strategy and we were to deep the proteome with a this we of the peptide in of the of HeLa the software with at both and protein in is a between the of peptide and the quantification of the time for a To we a and a method as in the 21 samples per the proteome coverage high for both with more than quantified proteins from more than This to unique proteins per gradient the the method in of with a of peptide and protein the and the the of proteins between and with and proteins in The method performed with to protein quantification with proteins with a than in the in the the of the proteome by data close to that the by the Evosep One with for high-throughput and acquisition of proteomic the great in high sensitivity nano-flow MS-based the robustness and throughput have weak even in of the MS-based proteomic This to a with a clinical at the of sensitivity M. I. C.L. proteomics sample preparation for mass Proteome 2018; 17: PubMed Scopus Google Scholar). we have an concept on the of at high-flow and This gradient the and is a column by a high-pressure pump. on these we a system that into a system - the Evosep established that of the by of the at the of the analytical column, chromatographic of the with the as a disposable sample the system is for sensitivity, and robustness - for clinical To we performed thousands of with cell as as complex clinical samples as that the of gradient with a system and the high-pressure peptide separation and operation without issues in chromatographic from the Evosep One to have and high of label-free quantitation injections.