FlashPack: Fast and Simple Preparation of Ultrahigh-performance Capillary Columns for LC-MS*

Capillary ultrahigh-pressure liquid chromatography (cUHPLC) is essential for in-depth characterization of complex biomolecule mixtures by LC-MS. We developed a simple and fast method called FlashPack for custom packing of capillary columns of 50–100 cm length with sub- 2 μm sorbent particles. FlashPack uses high sorbent concentrations of 500–1,000 mg/ml for packing at relatively low pressure of 100 bar. Column blocking by sorbent aggregation is avoided during the packing by gentle mechanical tapping of the capillary proximal end by a slowly rotating magnet bar. Utilizing a standard 100-bar pressure bomb, Flashpack allows for production of 15–25 cm cUHPLC columns within a few minutes and of 50 cm cUHPLC columns in less than an hour. Columns exhibit excellent reproducibility of back-pressure, retention time, and resolution (CV 8.7%). FlashPack cUHPLC columns are inexpensive, robust and deliver performance comparable to commercially available cUHPLC columns. The FlashPack method is versatile and enables production of cUHPLC columns using a variety of sorbent materials. Capillary ultrahigh-pressure liquid chromatography (cUHPLC) is essential for in-depth characterization of complex biomolecule mixtures by LC-MS. We developed a simple and fast method called FlashPack for custom packing of capillary columns of 50–100 cm length with sub- 2 μm sorbent particles. FlashPack uses high sorbent concentrations of 500–1,000 mg/ml for packing at relatively low pressure of 100 bar. Column blocking by sorbent aggregation is avoided during the packing by gentle mechanical tapping of the capillary proximal end by a slowly rotating magnet bar. Utilizing a standard 100-bar pressure bomb, Flashpack allows for production of 15–25 cm cUHPLC columns within a few minutes and of 50 cm cUHPLC columns in less than an hour. Columns exhibit excellent reproducibility of back-pressure, retention time, and resolution (CV 8.7%). FlashPack cUHPLC columns are inexpensive, robust and deliver performance comparable to commercially available cUHPLC columns. The FlashPack method is versatile and enables production of cUHPLC columns using a variety of sorbent materials. Capillary liquid chromatography (LC) is the central analytical separation technique for liquid chromatography-mass spectrometry (LC-MS)-based functional proteomics in biology, biomedicine, and clinical medicine. Combined with optimized and often multidimensional sample prefractionation, the LC-MS approach allows very detailed characterization of the human proteome. However, prefractionation comes at the cost of extended analysis time due to the need for LC-MS processing of each individual fraction (1Bekker-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 Syst. 2017; 4: 587-599.e4Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). The effectiveness of LC-MS itself can be improved by the application of ultrahigh-performance capillary chromatography (cUHPLC) 1The abbreviations used are:cUHPLCcapillary ultrahigh-performance liquid chromatographyEICextracted ion current chromatogramESIelectrospray ionizationFWHMfull width at half maximumIDinternal diameterLCliquid chromatographyLC-MSliquid chromatography-mass spectrometryTICtotal ion current chromatogram. 1The abbreviations used are:cUHPLCcapillary ultrahigh-performance liquid chromatographyEICextracted ion current chromatogramESIelectrospray ionizationFWHMfull width at half maximumIDinternal diameterLCliquid chromatographyLC-MSliquid chromatography-mass spectrometryTICtotal ion current chromatogram.. For example, analyte separation using a 50 cm length capillary column packed with 2 μm sorbent particles allowed profiling of the yeast proteome (∼5,000 proteins) in just about 1 h (2Hebert A.S. Richards A.L. Bailey D.J. Ulbrich A. Coughlin E.E. Westphall M.S. Coon J.J. The One Hour Yeast Proteome.Mol. Cell. Proteomics. 2014; 13: 339-347Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Further development of ultrahigh-performance and high-sensitivity LC separations is very important (3Shishkova 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 Scopus (93) Google Scholar) but is hampered by the fact that only few types of commercial cUHPLC columns are available, the choices of sorbents are limited, and commercial capillary columns come at high costs. A method for efficient, fast, and simple custom preparation of 50–100 cm capillary columns for robust and reproducible cUHPLC applications could change this situation. capillary ultrahigh-performance liquid chromatography extracted ion current chromatogram electrospray ionization full width at half maximum internal diameter liquid chromatography liquid chromatography-mass spectrometry total ion current chromatogram. capillary ultrahigh-performance liquid chromatography extracted ion current chromatogram electrospray ionization full width at half maximum internal diameter liquid chromatography liquid chromatography-mass spectrometry total ion current chromatogram. The most popular setup for capillary column packing today consists of a container (a 2 ml vial) with a stirred sorbent suspension placed into a special pressure bomb, which is connected to a nitrogen gas tank (Fig. 1A) (4Detailed explanation on the workings of the pressure bomb packing setup. Available from: https://www.nextadvance.com/pressure-injection-cells-lc-ms-capillary-column-packing-loader/?target=Overview.Google Scholar). Upon pressurization, the sorbent suspension is squeezed into the capillary open end dipped into the suspension. The sorbent is trapped and retained by a frit in the distal capillary end, i.e. a glass frit (5Cortes H.J. Pfeiffer C.D. Richter B.E. Stevens T.S. Porous ceramic bed supports for fused silica packed capillary columns used in liquid chromatography.J. High Resolution Chromatogr. 1987; 10: 446-448Crossref Scopus (138) Google Scholar) or a self-assembling sorbent frit in a tapered column end (6Ishihama 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 (259) Google Scholar). Capillary column packing is usually accomplished by low-pressure packing using a 100 bar pressure bomb and low sorbent concentration (2.5–25 mg/ml) (7Yates 3rd, J.R. McCormack A.L. Link A.J. Schieltz D. Eng J. Hays L. Future Prospects for the Analysis of Complex Biological Systems Using Micro-Column Liquid Chromatography-Electrospray Tandem Mass Spectrometry.Analyst. 1996; 121: 65R-76RCrossref PubMed Scopus (81) Google Scholar, 8Wang F. Dong J. Ye M. Wu R. Zou H. Integration of Monolithic Frit into the Particulate Capillary (IMFPC) Column in Shotgun Proteome Analysis.Anal. Chim. Acta. 2009; 652: 324-330Crossref PubMed Scopus (8) Google Scholar, 9Liu H. Finch J.W. Lavallee M.J. Collamati R.A. Benevides C.C. Gebler J.C. Effects of Column Length, Particle Size, Gradient Length and Flow Rate on Peak Capacity of Nano-Scale Liquid Chromatography for Peptide Separations.J Chromatogr. A. 2007; 1147: 30-36Crossref PubMed Scopus (64) Google Scholar). The approach is very popular due to its simplicity and is widely used in proteomics LC-MS laboratories. However, the low-pressure/low-concentration method is extremely time consuming and practically unsuitable for packing of very long (50–100 cm) capillary UHPLC columns (supplemental Fig. S1). Efficient and very fast cUHPLC column packing can be achieved at high pressures of 1,000–4,100 bar with high sorbent concentrations (100–250 mg/ml). However, this approach is technically more demanding due to the ultrahigh-pressure conditions (10MacNair J.E. Lewis K.C. Jorgenson J.W. Ultrahigh-Pressure Reversed-Phase Liquid Chromatography in Packed Capillary Columns.Anal. Chem. 1997; 69: 983-989Crossref PubMed Scopus (571) Google Scholar, 11Jorgenson J.W. Capillary Liquid Chromatography at Ultrahigh Pressures.Annu. Rev. Anal. Chem. 2010; 3: 129-150Crossref PubMed Scopus (93) Google Scholar, 12Bruns S. Franklin E.G. Grinias J.P. Godinho J.M. Jorgenson J.W. Tallarek U. Slurry Concentration Effects on the Bed Morphology and Separation Efficiency of Capillaries Packed with Sub-2 μm Particles.J. Chromatogr. A. 2013; 1318: 189-197Crossref PubMed Scopus (56) Google Scholar). We hypothesized that a simple and fast packing method can be developed using the low-pressure/high sorbent concentration combination provided that clogging and blocking of the column entrance by sorbent aggregation can be avoided during the packing process (supplemental Fig. S1A). We developed an optimized FlashPack approach, which uses a standard pressure bomb at 100 bar to achieve ∼100 times higher column packing rates than before and approaches the efficiency of ultrahigh-pressure packing protocols. The FlashPack protocol is versatile and simple to implement, and the produced cUHPLC columns exhibit performance comparable to that of similar commercially available columns. All chemicals and solvents were of LC gradient or LC-MS grade (Sigma); polyimide-coated fused silica capillary (360 μm outer diameter, 30–200 μm ID) was from PostNova. Chromatographic sorbent Inertsil ODS3 2 μm (GLSciences) was used in all LC-MS experiments shown here; other tested chromatography resins included Inertsil ODS3 3 μm (GLSciences), Reprosil Pur C18AQ 1.9, 3, and 5 μm (Dr. Maisch); BEH C18 1.7 μm (Waters); Aeris Peptide 2.6 μm and 1.7 μm; Luna 2 C18 3 μm; Luna Omega C18 1.6 μm (Phenomenex); Zorbax SB-C18 1.8 μm (Agilent); Triart C18 1.9 μm (YMC); PolyHYDROXYETHYL A 3 μm; and polyCAT A 2 μm (polyLC, Inc.). The EASY-Spray™ PepMap RSLC C18 2 μm (50 cm × 75 μm) column was obtained from Thermo Scientific. The pressure bomb was from Proxeon, Denmark. Similar pressure bomb devices (pressure injection cell, capillary packing unit) are also available from Next Advance and Nanobaume. Precut and polished fused silica capillaries were flushed with methanol and dried with N2 flow. Emitters for taper-tip columns were pulled with a P2000 laser puller (Sutter) according to the manufacturer's instructions. ESI emitters were visually inspected under a microscope. ESI needle tip diameter for 75 μm ID/360 mm outer diameter fused silica capillary was ∼5 μm (proportionally smaller and larger for other capillary IDs). Integrated frits for fritted capillary columns were made using glass-paper (GF/C, Whatman) soaked in Kasil-formamide mixture (13Maiolica A. Borsotti D. Rappsilber J. Self-Made Frits for Nanoscale Columns in Proteomics.Proteomics. 2005; 5: 3847-3850Crossref PubMed Scopus (80) Google Scholar). Columns were packed into prepared fused silica fritted/tapered capillaries in a packing bomb pressurized to 100 bar with N2. Sorbent suspension was prepared in methanol in a 2 ml flat-bottom glass vial (VWR Cat. No. 66020–950). The required quantity of the sorbent (see below) was added to the vial and mixed with methanol using a vortexer. Sorbent was left to swell for 30 min and then vortexed and sonicated in an ultrasonication bath for 60 s. The sorbent suspension vial was placed inside the pressure bomb. The capillary (fritted or tapered) was mounted with its open end (proximal end) dipped into the vial, and fixed with a top nut. The pressure bomb system was pressurized to 100 bars by N2 gas. The capillary column was packed to a length that was 5 to 10 cm longer than the desired final column length. After packing, the pressure was slowly released over a 10 min period to avoid bubbling inside the column. The freshly packed capillary column was connected to and run on an LC system with 95% acetonitrile at 150 nl/min flow rate (75 μm ID; for other IDs recalculate the flow rate proportionally to the capillary cross section) for 30 min to compress the sorbent bed. The column was then cut to the desired length. Capillary columns were stored fully immersed in 10% aqueous ethanol. A volume of 400 μl low-concentration sorbent suspension (2–50 mg/ml) in methanol was prepared as described above. A magnet bar (2 × 3 mm) was put into the vial and rotated at 1,000–1,500 rpm to keep the sorbent evenly suspended during the packing process. The capillary was mounted in a pressure bomb with the open proximal end positioned 4–5 mm above the bottom of the vial with the sorbent suspension, without touching the rotating magnet. The rest of the protocol is as described above. 50–100 mg of sorbent (a 2–3 mm layer of dry sorbent in a flat-bottomed vial) was resuspended in 1 ml of methanol. The sorbent was left to settle by gravity for 20–30 min. The final settled sorbent particle layer on the bottom of the vial must be at least 5 mm deep. The vortex–sonication–settling cycle must be repeated if the sorbent suspension was not in use for more than 12 h. A small magnet stirring bar (2 × 3 mm) was placed in the sorbent vial and set to rotate at a low speed (400–500 rpm). An empty capillary (no solvent inside) was mounted in the packing bomb so that it reached more than 2 mm below the surface of the settled sorbent layer and 1–2 mm above the bottom of the vial. To achieve that, the capillary was pushed all the way to the bottom of the vial then retracted 1–2 mm and fixed with a nut. The capillary and the magnet bar must contact each other for the whole packing time, so the rotating magnet bar provides continuous mechanical tapping of the open proximal capillary end. The bomb was pressurized immediately after mounting the capillary in order to avoid passive filling of the capillary with the solvent. The packing process was visually controlled. If the mechanical tapping of the proximal capillary end (cupola destabilization) was not effective, there was no appearance of opaque sorbent slurry being transferred into the capillary column—the capillary remained filled with the solvent and was fully transparent. In this case, the magnet bar speed was to If the packing not the pressure bomb was and the capillary end The of the packing was as described in the above. or human were at in 50 mm 10 mm mm with a were using an in concentration was by J.R. and and Peptide for Analysis.Anal. Chem. PubMed Scopus Google Scholar) and to 1 mg/ml with were into of and at for 10 min. of were and the were at and at The was with and dried for 10 min. 100 μl of in 50 mm were and the sample was for 5 min at to the was using 1 of 100 of for 5 h at an of 100 of was and the sample was at The was using 10 μl of 10% concentration was by M. Y. for Proteome Proteome PubMed Scopus Google Scholar, of Efficiency for Cell. Proteomics. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). Peptide concentration was using J.R. and and Peptide for Analysis.Anal. Chem. PubMed Scopus Google Scholar). were stored at to LC-MS were and sonicated for 1 min and for min at LC-MS analysis was using an system connected to a Capillary columns were using a (50 in a min LC were on PepMap 100 C18 5 μm × 5 mm in solvent at 10 flow and with a gradient of in from to of solvent in 2 h or or from 2 to in min at flow. Separation was with a FlashPack column packed with Inertsil sorbent or with a commercially available 75 μm × 50 cm PepMap 100 2 μm column the and the column were at 50 cm or at cm An developed was used for FlashPack columns. Mass spectrometry were in with 2 cycle were in an and were in a ion were as injection time 50 × were with 1.6 the of to and of 5 × was set to 30 s. was in with were for with 3 × for all time was were in rate and in ESI was 2 for columns and for 50 cm and for cm FlashPack were using M. R. M.J. An for and Proteomics 2010; PubMed Scopus Google Scholar) and Peptide and was using J. Mann M. High Peptide Mass and PubMed Scopus Google Scholar) and J. L. M. D. for and Peptide Cell. Proteomics. Full Text Full Text PDF Scopus Google Scholar). In the were 2016; was used for detailed LC analysis time, and and for was using the and as and for and Peptide and The were and All were as only by not by time and width width and are from the was used for analysis using and The Proteome was set to 5 and ion to The were fixed and with rate was using the was only for (no with retention time min and using total ion current All were as were with 2 and 2 and were to was using the full gradient time and width at there is no available proteomics of for complex with of used full width at half which is to × Chromatographic and were by processing in a after in A was in and over and were as full width at of width at of to the left from time, and a and are to the left and to the of the time at 10% of Column and reproducibility were on the of retention time width width and and of of reproducibility of and The of this was to a technically low-pressure protocol for fast packing of long UHPLC capillary columns. To achieve it was to capillary entrance blocking by sorbent clogging during column packing at high sorbent concentrations (supplemental Fig. S1A). We that the blocking must be than of a of sorbent blocking the entrance a there must be a self-assembling that of a or in and R. M. Scholar) and not to a self-assembling frit in a pulled (6Ishihama 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 (259) Google Scholar) (supplemental Fig. The sorbent be by sorbent particle aggregation and solvent flow in the way and a self-assembling is for but it sorbent from into the column packing (Fig. the solvent flow is essential for the the self-assembling at the capillary entrance be produced or the pressure bomb and be However, a very similar blocking process capillaries of the larger capillary is being the smaller capillary is to the sorbent by a self-assembling blocking (supplemental Fig. We hypothesized that continuous of the packing at very high sorbent concentrations of system FlashPack method mechanical with column packing from sorbent suspension is by mechanical of the proximal capillary end by a small magnet bar. The capillary must be positioned in the sorbent slurry vial that the open end of the capillary the rotating magnet which and and of sorbent that with the column packing (Fig. Fig. and approaches are also in the FlashPack method (supplemental The column packing rate is to the sorbent suspension The concentration can be achieved in the of a gravity settled sorbent the concentration can as high as 500–1,000 mg/ml (supplemental The of the packing process is shown on Fig. The capillary is mounted inside the vial with the settled sorbent layer Upon system the capillary filled with the sorbent slurry magnet bar and and allows for continuous packing to the desired column length The speed of the magnet is to a speed that is for (400–500 rpm). High speed to sorbent by a magnet bar and on the of and Capillary Columns.Anal. Chem. 2017; PubMed Scopus Google Scholar) and the settled sorbent the concentration and packing the the sorbent suspension inside the column can be with the as by (see Fig. An empty capillary filled with a sorbent suspension without mechanical (supplemental Fig. We that the very high flow rate can the by itself in a way a under higher The column entrance blocking was for packing, which at proportionally higher flow rates than the low-pressure However, as the capillary filled with the liquid and the to packed at the distal end of the the the flow rate and the If no is no sorbent can and the solvent flow the sorbent inside the column capillary (supplemental Fig. In a column can be produced in a very time with no mechanical The of the sorbent inside the capillary during the filling is to the capillary so that the capillary length the desired column length by For example, a 50 cm capillary is for packing 15–25 cm and cm capillaries for packing 50 cm columns using the FlashPack The FlashPack protocol the packing speed and the packing time by 100 (a few for standard capillary columns of cm length. The time required to a 50 cm FlashPack cUHPLC column with 2 μm of sorbent is less than 1 h (Fig. and The FlashPack packing was tested with types of fully silica sorbents Luna Luna as as and and liquid chromatography and sorbents A and polyCAT not The of the tested sorbent particles from 1.6 to 5 The FlashPack approach for all tested sorbents for Triart which required less as or for the sorbent suspension. filling of the capillary with the sorbent suspension (supplemental Fig. 2 and is not by the sorbent the suspension the columns can be filled with 5 1.6 or sub- 1 μm sorbents in We tested the FlashPack protocol with fused silica capillaries of from 30 to μm not FlashPack was for fritted capillary columns and pulled capillary columns for to ESI We that column packing at high sorbent slurry concentration improved the efficiency of frit in the tapered column end, the rate of column blocking immediately after the bomb The columns produced with the FlashPack approach excellent separation and A 50 cm FlashPack column was used over a period for proteomics sample analysis for more than sample performance analysis to a commercial column with similar and sorbent was before and after (supplemental Fig. the the FlashPack column was and to the LC The FlashPack column separation comparable to the commercial column immediately after the packing and after use without (Fig. Fig. The column excellent reproducibility and during a proteomics of (supplemental Fig. The proteomics obtained using FlashPack columns were in with J. A. Proteome during of Human Proteome PubMed Scopus Google Scholar). that column packing from high sorbent slurry concentrations not resolution S. Franklin E.G. Grinias J.P. Godinho J.M. Jorgenson J.W. Tallarek U. Slurry Concentration Effects on the Bed Morphology and Separation Efficiency of Capillaries Packed with Sub-2 μm Particles.J. Chromatogr. A. 2013; 1318: 189-197Crossref PubMed Scopus (56) Google Scholar, and on the of and Capillary Columns.Anal. Chem. 2017; PubMed Scopus Google Scholar, Godinho J.M. Jorgenson J.W. Tallarek U. Bed with an Slurry Concentration for of Efficient Capillary Ultrahigh Liquid Chromatography Chromatogr. A. 2017; PubMed Scopus Google Scholar). To the reproducibility and analytical performance of FlashPack column a was using cm long and 75 μm FlashPack ESI columns (Fig. The resolution 50 and cm capillary column to the M. M. Gebler J.C. of Column Peak Capacity on the Separation of Complex Peptide in and Liquid Chromatogr. A. PubMed Scopus Google The full width was on improved from to and the of IDs LC-MS run from to from (supplemental Fig. The standard the tested columns was which was comparable to that for ultrahigh-pressure packing J.M. Tallarek U. Jorgenson J.W. of High Slurry Concentration and to Capillary Ultrahigh Liquid Chromatography Chromatogr. 2016; PubMed Scopus Google Scholar). The FlashPack approach cUHPLC columns for enables to experiments with ultrahigh-performance column chromatography using sorbents and column for applications of cUHPLC and LC-MS in and The FlashPack method uses a standard pressure bomb that is in LC-MS and can be