A New Parallel High-Pressure Packing System Enables Rapid Multiplexed Production of Capillary Columns

Abstract

•We present a newly designed high-pressure packing station for capillary columns.•Detailed part lists and manufacturing instructions included.•40 to 800 times faster packing of capillary columns for high-performance proteomics.•Columns produced with the presented setup have state-of-the-art quality metrics. Reversed-phase HPLC is the most commonly applied peptide-separation technique in MS-based proteomics. Particle-packed capillary columns are predominantly used in nanoflow HPLC systems. Despite being the broadly applied standard for many years, capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column packing with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as the driving fluid, our system replaces the traditional setup of helium-pressured packing bombs. By using 10× multiplexing, we have reduced the production time to just under 2 min for several 50 cm columns with 1.9-µm particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters and lengths packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station. Reversed-phase HPLC is the most commonly applied peptide-separation technique in MS-based proteomics. Particle-packed capillary columns are predominantly used in nanoflow HPLC systems. Despite being the broadly applied standard for many years, capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column packing with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as the driving fluid, our system replaces the traditional setup of helium-pressured packing bombs. By using 10× multiplexing, we have reduced the production time to just under 2 min for several 50 cm columns with 1.9-µm particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters and lengths packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station. State-of-the-art MS-based proteomic pipelines typically consist of a sample preparation workflow to digest proteins and harvest pure peptides, an LC system for peptide separation, a mass spectrometer, and a sophisticated bioinformatics pipeline for raw data interpretation and subsequent statistical analysis (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5271) Google Scholar, 2Aebersold R. Mann M. Mass-spectrometric exploration of proteome structure and function.Nature. 2016; 537: 347-355Crossref PubMed Scopus (848) Google Scholar). The LC system plays a central role by partially separating the complex mixture of tens of thousands of peptides in a time-resolved manner according to their physicochemical properties, making them ultimately manageable for the MS system over the course of a gradient (3Michalski A. Cox J. Mann M. More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS.J. Proteome Res. 2011; 10: 1785-1793Crossref PubMed Scopus (431) Google Scholar, 4Shishkova E. Hebert A.S. Coon J.J. Now, more than ever, proteomics needs better chromatography.Cell Syst. 2016; 3: 321-324Abstract Full Text Full Text PDF PubMed Google Scholar). The most widely applied technique for high-performance applications is reversed-phase separation, originally introduced in the 1970s (5Horváth C. Melander W. Molnár I. Solvophobic interactions in liquid chromatography with nonpolar stationary phases.J. Chromatogr. A. 1976; 125: 129-156Crossref Scopus (1219) Google Scholar). In essence, chromatographic systems are made of programmable pumps with the ability to form a gradient of a mixture of different agents. In the case of reversed-phase LC, the stationary phase is nonpolar, separating analytes by hydrophobicity over the course of a gradient of an increasing nonpolar mobile phase. The LC system is coupled to the mass spectrometer by electrospray (ES) ionization via an emitter (6Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Google Scholar). Glass or steel needles are commonly connected to the column. Particle-packed capillaries for chromatography can also be used for ES without being coupled to an additional emitter (7Kennedy R.T. Jorgenson J.W. Preparation and evaluation of packed capillary liquid chromatography columns with inner diameters from 20 to 50 micrometers.Anal. Chem. 1989; 61: 1128-1135Crossref Scopus (318) Google Scholar, 8Emmett M.R. Caprioli R.M. Micro-electrospray mass spectrometry: Ultra-high-sensitivity analysis of peptides and proteins.J. Am. Soc. Mass Spectrom. 1994; 5: 605-613Crossref PubMed Scopus (465) Google Scholar, 9Ishihama Y. Rappsilber J. Andersen J.S. Mann M. Microcolumns with self-assembled particle frits for proteomics.J. Chromatogr. A. 2002; 979: 233-239Crossref PubMed Scopus (241) Google Scholar). These basic attributes are shared by most LC-MS systems, and differences are mainly defined by operational flow. Nanoflow LC operates at flow rates of several hundred nanoliters per minute and is the standard in proteomics because of the high sensitivity obtainable. High flow rates in the μl to ml range, applied to columns with large inner diameters (IDs), are typically used in high-throughput or industrial-scale analysis and analytical MS application areas. Although these microflow and analytical-flow systems limit sensitivity, recent work has demonstrated robust and reproducible performance (10Bian Y. Zheng R. Bayer F.P. Wong C. Chang Y.C. Meng C. Zolg D.P. Reinecke M. Zecha J. Wiechmann S. Heinzlmeir S. Scherr J. Hemmer B. Baynham M. Gingras A.C. et al.Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS.Nat. Commun. 2020; 11: 157Crossref PubMed Scopus (62) Google Scholar, 11Bian Y. Bayer F.P. Chang Y.C. Meng C. Hoefer S. Deng N. Zheng R. Boychenko O. Kuster B. Robust microflow LC-MS/MS for proteome analysis: 38 000 runs and counting.Anal. Chem. 2021; 93: 3686-3690Crossref PubMed Scopus (6) Google Scholar). Reproducibility and stability of those systems are high, but drawbacks are lowered sensitivity and a need for high sample amounts. Compared with developments in sample preparation, MS instrumentation, scan modes, and software, the LC apparatus has been largely unchanged in cutting-edge MS-based proteomics. Although identifications in proteomics experiments have doubled in single-shot experiments, this can mainly be traced to improvement on the MS instrumentation and software (12Bernhardt O. Selevsek N. Gillet L. Rinner O. Picotti P. Aebersold R. Reiter L. Spectronaut: a fast and efficient algorithm for MRM-like processing of data independent acquisition (SWATH-MS) data.F1000Res. 2014; 51092Google Scholar, 13Cox 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 (7561) Google Scholar, 14Hu Q. Noll R.J. Li H. Makarov A. Hardman M. Graham Cooks R. The Orbitrap: A new mass spectrometer.J. Mass Spectrom. 2005; 40: 430-443Crossref PubMed Scopus (921) Google Scholar, 15Kelstrup 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 (123) Google Scholar, 16Meier F. Beck S. Grassl N. Lubeck M. Park M.A. Raether O. Mann M. Parallel accumulation-serial fragmentation (PASEF): Multiplying sequencing speed and sensitivity by synchronized scans in a trapped ion mobility device.J. Proteome Res. 2015; 14: 5378-5387Crossref PubMed Scopus (108) Google Scholar, 17Meier F. Brunner A.D. Frank M. Ha A. Bludau I. Voytik E. Kaspar-Schoenefeld S. Lubeck M. Raether O. Bache N. Aebersold R. Collins B.C. Röst H.L. Mann M. diaPASEF: parallel accumulation–serial fragmentation combined with data-independent acquisition.Nat. Methods. 2020; 17: 1229-1236Crossref PubMed Scopus (26) Google Scholar). Current trends in LC developments aim rather toward systems for higher throughput and increasing robustness required for clinical applications (18Bache N. Geyer P.E. Bekker-Jensen D.B. Hoerning O. Falkenby L. Treit P.V. Doll S. Paron I. Müller J.B. Meier F. Olsen J.V. Vorm O. Mann M. A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics.Mol. Cell Proteomics. 2018; 17: 2284-2296Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), whereas the race for better separation in single-shot high performance runs with increasingly higher pump pressures has been comparatively abandoned. Consequently, a typically used setup for maximum sensitivity and performance for most experiments still consists of columns around 75-μm ID with a length of 20 to 50 cm, packed with sub-2-μm particles. Although, better performance could be reached by longer columns or smaller particles, both conditions would result in higher operational pressures that tend to make the LC systems unstable (4Shishkova E. Hebert A.S. Coon J.J. Now, more than ever, proteomics needs better chromatography.Cell Syst. 2016; 3: 321-324Abstract Full Text Full Text PDF PubMed Google Scholar, 19Richards A.L. Merrill A.E. Coon J.J. Proteome sequencing goes deep.Curr. Opin. Chem. Biol. 2015; 24: 11-17Crossref PubMed Scopus (64) Google Scholar). For example, very high pressures can lead to leaks in the LC flow paths, resulting in poor reproducibility and subsequently a loss of measurement time. Commercially available capillary columns in the aforementioned dimensions are expensive, especially considering how frequently they must be replaced (e.g., in our laboratories, a 50 cm column with 75-μm ID has an average turnaround time from 10 to 14 days). Therefore, many high-throughput laboratories produce packed capillaries in-house. Empty glass capillaries, ready to be packed and used, can be either purchased or produced from cheap polyimide-coated capillaries using a laser puller. Typically, a gas pressure system is deployed to pack such columns with particles in the low μm range, and instructions on the manufacturing process can be found online with open access (https://proteomicsresource.washington.edu/docs/protocols05/Packing_Capillary_Columns.pdf). However, this process is inherently slow, and interesting methods have recently been established with the aim of speeding up the packing process with high pressure (20Shishkova E. Hebert A.S. Westphall M.S. Coon J.J. Ultra-high pressure (>30,000 psi) packing of capillary columns enhancing depth of shotgun proteomic analyses.Anal. Chem. 2018; 90: 11503-11508Crossref PubMed Scopus (22) Google Scholar) or dense bead slurry, as in the FlashPack method (21Kovalchuk S.I. Jensen O.N. Rogowska-Wrzesinska A. FlashPack: Fast and simple preparation of ultrahigh-performance capillary columns for LC-MS.Mol. Cell Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). these we present a high-pressure packing system for capillary columns using a bead that has been as for column performance S. Jorgenson J.W. on the and separation of capillaries packed with μm Chromatogr. A. PubMed Scopus Google Scholar). These high and packing pressures of to to achieve packing times for 50 cm columns in the minute with our with for traditional a system and a pump of high flow rates packing to up to columns and column production 40 to 800 times more time efficient than in systems. We column performance for packing pressures at over with on the column and packing times to at higher We a of the system can be up in laboratories from for 75-μm for or for to by a and the with an in the of the capillary at a of 2 ES emitter with a laser at the part of the capillary resulting in capillary columns ready to be in with and from using a and of with and at preparation with the We used with per the peptide peptide via and the peptide to using LC-MS instrumentation of an ultra-high-pressure system coupled to an using a ion peptides on high-pressure packed columns as in the and For LC-MS/MS analysis with 75-μm ID peptides For ID peptides used, and for ID peptides used to for the higher column in and with a min gradient of to of by a 10 min to of and a min of B. For the 75-μm ID the flow for ID and for ID columns to for flow The column at by an in-house a and in time by the MS data with a data-dependent scan to in the to with a maximum time of and a of at of by with a of scans at a of at with an of and a maximum time of to to sequencing of column with min runs the for column MS raw by MaxQuant software, and peptide lists the A by the with as a and and as We the to for protein and peptide with a length of for peptides, and the by a as to and as using and as A maximum of identification with an mass up to and a mass of 20 proteins and peptides to the in using and The on making different capillary columns for proteomics experiments as as achieve statistical analysis from experiments for the packing time and pressure performance conditions for column to on LC and MS systems and A central of chromatography in proteomics laboratories is the for new capillary columns. to their columns be as a However, in our we frequently performance only for a short for Therefore, to the and we and many laboratories produce own capillary columns. However, the throughput of production is especially for columns with a ID and length such as the 50 cm 75-μm ID columns used in most applications in our We produce or capillaries and pack them with phase typically sub-2-μm A can of columns a and columns are also to However, the packing process is inherently and high-performance columns in MS In the of longer column lengths is our a for We that high-throughput packing of capillary columns could be by bead (21Kovalchuk S.I. Jensen O.N. Rogowska-Wrzesinska A. FlashPack: Fast and simple preparation of ultrahigh-performance capillary columns for LC-MS.Mol. Cell Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) in combination with packing (20Shishkova E. Hebert A.S. Westphall M.S. Coon J.J. Ultra-high pressure (>30,000 psi) packing of capillary columns enhancing depth of shotgun proteomic analyses.Anal. Chem. 2018; 90: 11503-11508Crossref PubMed Scopus (22) Google Scholar). However, an packing pressure and bead can lead to column and the packing as a bead as an to this because can higher bead However, in combination with our bead particles, we poor chromatographic performance proteomic we combined packing pressure with the FlashPack system (21Kovalchuk S.I. Jensen O.N. Rogowska-Wrzesinska A. FlashPack: Fast and simple preparation of ultrahigh-performance capillary columns for LC-MS.Mol. Cell Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar), bead at the column via our we a for high-pressure the pressure from a conventional HPLC system in our The consists of a central the bead and and has A access the with the bead slurry, a the capillary the and a is used as an for the pressure from the HPLC system The applied to pack columns in this system can be the slurry, we of bead particles with of and minute of in a we the for we of the with a The packing to single capillaries using the HPLC high-pressure pumps However, this system for high-throughput column and the low pump of the HPLC system in packing as the pump to be several times a column with by of our newly packing we to column We replaced the HPLC pump with a This system driving gas from a standard gas supply at a pressure of to a with a pressure of and flow of the FlashPack we used methanol as the packing at the The high flow to pump for packing of up to columns with our station. We the packing to high-pressure For we a system with on via to the packing in and we connected a high-pressure to packing pressure and a pressure for efficient and of the a Although the system is typically at in our the of pressure only without from the from the In the system is from driving gas pressure by a the pump to be to a higher than bar. with conventional packing systems, the is the of the capillary to the high-pressure We used a standard used in HPLC applications in combination with a newly designed, to the column under very high the system pressure the of the the column is to the low of this is has the a and this must be Compared with can more than from our new packing station. and we up the packing system in a with to pump methanol or bead particle and the for The time required to a capillary column with on the bead of the packing and the flow the Empty capillaries with a ES emitter have high flow rates in the for conventional packing with pressure However, as the bead the flow the column the bead of FlashPack enables short packing times especially for columns (21Kovalchuk S.I. Jensen O.N. Rogowska-Wrzesinska A. FlashPack: Fast and simple preparation of ultrahigh-performance capillary columns for LC-MS.Mol. Cell Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). We that this with the high flow rates of our high-pressure system would packing times. the production throughput of our we packed capillaries with 75-μm ID at different pressures and the time a bead packing at the pressure on average pressure to in packing times just over a higher pressure result in faster our system the time for making a single column to with packing (20Shishkova E. Hebert A.S. Westphall M.S. Coon J.J. Ultra-high pressure (>30,000 psi) packing of capillary columns enhancing depth of shotgun proteomic analyses.Anal. Chem. 2018; 90: 11503-11508Crossref PubMed Scopus (22) Google Scholar, S.I. Jensen O.N. Rogowska-Wrzesinska A. FlashPack: Fast and simple preparation of ultrahigh-performance capillary columns for LC-MS.Mol. Cell Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) A and the production throughput is higher to packing and the to capillaries and bead This in a of 40 to 800 with bead and on the high-pressure the packing can be used to pack several columns This requires the system via the pressure and the columns with packing of several columns from the the packing speed because of the of from the packing the bead has to be and 10 min for Typically, we the capillary The average for columns is 20 the production of of columns in a additional of the high-throughput system is that to packed in of The high-pressure system the as packing are particle the capillary and bead at the column can only be by This and of and the of and particles for bead from dense can be by conditions according to the FlashPack (21Kovalchuk S.I. Jensen O.N. Rogowska-Wrzesinska A. FlashPack: Fast and simple preparation of ultrahigh-performance capillary columns for LC-MS.Mol. Cell Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). the of packing pressure on column performance on we of our standard on column. packing we in the of peptides and protein the of peptides for conditions the times of peptides using columns produced at pressures high and from packed with pressure conditions used to column performance is the that can be as in A. A. Scholar). the at is used for but in proteomics experiments tens of thousands of are the is typically at maximum is also We to the at as a In the of than would be from an analysis of but the typically around the of The of the at for the and for higher packing pressures up to a of In the in the and are J. W. of mobile phase to of peptides in Chromatogr. PubMed Scopus Google Scholar). The this with the higher packing pressures could result from bead the performance for the proteomics to the that the in at with higher packing pressures is the LC-MS This in an only of experiments of columns packed at different pressures the of peptide is that for the peptides elute in a and reproducible time that is by the applied packing This time stability is by separation of the different can be by the at of analysis of peptides with at whereas the differences a toward a better performance for or higher packing pressures of capillary columns. We a in column from the different packing on these that the packing pressure has or only on the column The length and ID of capillary columns their to a of sample and LC systems, separation and In MS-based 75-μm ID columns in combination with flow rates in the of to per minute are we packed such capillary columns with different lengths 50 with our high-pressure system and their time for the columns faster and in the of The columns produced the and subsequently in the of peptides and proteins A and the of peptide and the at also and the years, the for high-throughput analysis has for the analysis of clinical especially as we have P.E. Mann M. by proteomics.Mol. Syst. Biol. PubMed Scopus Google Scholar). This has been by a novel HPLC with gradients and higher flow rates (18Bache N. Geyer P.E. Bekker-Jensen D.B. Hoerning O. Falkenby L. Treit P.V. Doll S. Paron I. Müller J.B. Meier F. Olsen J.V. Vorm O. Mann M. A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics.Mol. Cell Proteomics. 2018; 17: 2284-2296Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and by systems in the high per minute (10Bian Y. Zheng R. Bayer F.P. Wong C. Chang Y.C. Meng C. Zolg D.P. Reinecke M. Zecha J. Wiechmann S. Heinzlmeir S. Scherr J. Hemmer B. Baynham M. Gingras A.C. et al.Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS.Nat. Commun. 2020; 11: 157Crossref PubMed Scopus (62) Google Scholar, C. N. F. A. S. M. enables proteomics using chromatography and Scopus Google Scholar). these columns with a higher ID to pressure we produced columns with and ID and their column the setup has to be to the of capillaries different we the flow rates to the and the amount of to the column For the ID this in a flow of and of peptides for whereas for the ID and 2 of peptide to be to the and used for the 75-μm ID columns. This of higher sample amount the of column diameters for with The μl of pump from the used for the to a 2 gradient with the ID but longer gradients or higher flow rates would the of the LC system and flow The higher column to but peptide and protein identifications to the of the sample we a in the peptide and the at also by the column ID Here, we to the throughput and to the production of capillary columns for MS-based proteomics. We a for the and the of our high-pressure packing station. The setup can be at low with the for columns. We designed this new station to columns a the packing process of capillary columns more than a with traditional gas In this we our system by the and column production In the high pressures the packing of columns The ability to produce columns at high-throughput for the of only using capillary columns at the of their performance, them as as or ionization is in of robustness of the packing process and the stability at high we have in the performance over a of packing pressure from to 3000 bar. We the laboratories of to high-performance capillary columns.

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