Nano LC at 20 nl/min Made Easy: A Splitless Pump Combined with Fingertight UHPLC Nano Column to Boost LC-MS Sensitivity in Proteomics

Transcription

1 Nano LC at 20 nl/min Made Easy: A Splitless Pump Combined with Fingertight UHPLC Nano Column to Boost LC-MS Sensitivity in Proteomics Rieux L 1., De Pra M 1., Köcher T 2., Mechtler K 2., Swart R 1 1 Thermo Fisher Scientific, Amsterdam, The Netherlands 2 Research Institute of Molecular Pathology, Vienna, Austria

2 Overview Purpose: An easier and robust solution to nano LC-ESI- MS at ultra-low flow rates is demonstrated. Methods: 25 µm ID columns were operated with a splitless pump at flow rates lower than any existing commercial system. Results: Performance of the column and UHPLC system are excellent. Preliminary data on MS sensitivity are shown. Introduction Increased sensitivity in nano LC-ESI-MS is generally achieved by decreasing the inner diameter (ID) of the column, resulting in higher peak concentration and more ions detected with mass spectrometry. Consequently, more peptides can be successfully sequenced and thereby improve protein identification. The number of peptides detected in complex samples can increase by more than a factor of 100 just by decreasing the column diameter from 75 µm to 30 µm ID 1. With the reduction in column ID, the gradient flow delivered by the pump needs to be scaled down accordingly. For columns of 30 µm ID (or less), the required flow rates fall well below those of most commercially available nano-lc pumps. This was traditionally achieved by implementing a custom-made flow splitter in the commercial systems. In the present study, we used a splitless pump tailored to the flow requirements of peptide analysis using 25-µm ID columns. Methods. Liquid Chromatography Thermo Scientific Dionex UltiMate 3000 RSLCnano system Column: 25 µm ID x 15 cm, packed with Thermo Scientific Acclaim PepMap RSLC C18, 2 µm, 100 Å Trap: 50 µm ID x 7 cm (2 cm packed bed), packed with Acclaim PepMap C18, 3 µm, 100 Å Mass Spectrometry Thermo Scientific Q Exactive Mass Spectrometer New Objective FS D-5 Nano-ESI emitter Data Analysis Thermo Scientific Dionex Chromeleon 6.8 Chromatography Data System Results Column Performances The 25 µm ID nano columns were packed in silica capillary. Both column inlet and outlet were fritted according to a proprietary protocol. The columns were implemented with fingertight connections capable of withstanding 1000 bar without leaking. Each column was tested after production in a direct injection configuration with UV detection. Tryptic peptide mixtures were separated in gradient mode to evaluate the columns. Peak width and symmetry performances were excellent (Figure 1). 2 Nano LC at 20 nl/min Made Easy: A Splitless Pump Combined with Fingertight UHPLC Nano Column to Boost LC-MS Sensitivity in Proteomics

3 FIGURE 1. Direct injection of Cytochrome-C digest in a 25 µm ID nano column. UV detection. Solvent A: TFA 0.05%; Solvent B: 2/8 water/acn (0.04% TFA). 4à55 %B in 30 minutes at 50 nl/min. The peaks label is the width at half height. mau 5,4 sec 4,5 sec 4.6 sec 5.2 sec 4.9 sec 5.3 sec 7.6 sec 4.9 sec 4.3 sec 4.4 sec 4.6 sec min 49.5 Splitless Nano Pump Performances The UltiMate RSLCnano UHPLC system was adapted to meet the requirements of ultra-low nano flow. The flow selector was replaced with a prototype version suitable for controlling flow rates lower than 70 nl/min. The flow delivery precision of the pump was tested by evaluating the relative standard deviation (RSD) of the retention time of the peptide peaks. The precision was outstanding throughout the flow range used for the experiments (Table 1 and Table 2). The prototype flow selector can be easily and reversibly implemented in any UltiMate 3000 RSLCnano system. TABLE 1. Retention time precision of 6 peptides at 20 nl/min. The rest of the conditions are the same as Figure 1. Peak RT (min) RSD (RT, %) TABLE 2. Retention time precision of a selected peptide at various flow rates. Conditions are the same as Figure 1 except for the flow rate. Flow Rate (nl/min) RT (min) RSD (%) Pre-Concentration with Vented Column The plumbing of the system for a pre-concentration experiment can be seen in Figure 2. The pressure regulators are packed capillaries with specific flow resistance (similar to the nano column or trap column pressure). The purpose of the pressure regulator is to mantain the nano pump and the loading pump pressurization during the whole run, irrespective of the valve position. FIGURE 2. Plumbing schematic for vented column pre- Thermo Scientific Poster Note PN _e 3/12S 3

4 FIGURE 2. Plumbing schematic for vented column preconcentration. Even when pre-concentration was set up, the precision of the analysis was preserved. The flow restrictors were essential to ensure fast and repeatable pressurization after valve switching. FIGURE 3. Overlay of 6 consecutive injections with sample pre-concentration and 25 µm ID column. Sample: 300 fmol protein mixture digest (Cytochrome C, lysosyme, alcohol dehydrogenase, BSA, apo-transferrin, ß-galactosidase). Solvent A: TFA 0.05%; Solvent B: 2/8 Water/ACN (0.04% TFA). 4à55 %B in 60 minutes at 50 nl/min. Loading solvent TFA 0.1% at 2 µl/min. UV detection (214 nm) mau min The efficiency loss when using pre-concentration with the vented column was marginal (Figure 4). Using the same gradient and sample, the analysis of peak width (half height) for 2 hydrophilic and 2 hydrophobic peptides showed that the peak width increase is well below 1 second. 4 Nano LC at 20 nl/min Made Easy: A Splitless Pump Combined with Fingertight UHPLC Nano Column to Boost LC-MS Sensitivity in Proteomics

5 FIGURE 4. Comparison of the peak width at half height in direct injection and pre-concentration. Solvent A: TFA 0.05%; Solvent B: 2/8 Water/ACN (0.04% TFA). 4à55 %B in 30 min. at 50 nl/min. Loading solvent TFA 0.1% at 2 µl/min. UV detection (214 nm). 6 5,5 PWHH (sec) 5 4,5 direct injection Pre- concentration Preliminary UHPLC-UV-MS Results In the initial UHPLC-MS experiments, UV detection was used to monitor the chromatographic performances. The column was connected to a 0.75 nl flow cell with 10 µm ID silica capillaries, and the flow cell was connected to the emitter with 20 cm, 20 µm ID silica capillaries. FIGURE 5. The number of identified peptides for different gradient times using a 25 µm ID column. Sample: 0.1 µg Hela lisate. Solvent A: Formic acid 0.1%; Solvent B: 2/8 Water/ACN (0.1% formic acid) 4à55 %B at 50 nl/min. Loading solvent TFA 0.1% at 1.5 µl/min identified peptides Gradient time (h) FIGURE 6. The number of identified proteins for different gradient time using a 25 µm ID column. Sample: 0.1 µg Hela lisate. Solvent A: Formic acid 0.1%; Solvent B: 2/8 Water/ACN (0.1% formic acid) 4à55 %B at 50 nl/min. Loading solvent TFA 0.1% at 1.5 µl/min identified proteins Gradient time (h) Conclusion Thermo Scientific Poster Note PN _e 3/12S 5

6 Gradient time (h) Conclusion High performance, user friendly 25 µm ID columns packed with 2 µm C18 stationary phase were developed and tested. The Ultimate 3000 RSLCnano was configured to efficiently deliver flow between 20 and 50 nl/min without splitting. Retention time precision was excellent between 20 and 50 nl/min. More than 12 thousand peptides could be identified for a 5 hour run time at 50 nl/min. Currently we are evaluating MS sensitivity at 20 nl/min and improving the interfacing between the column and the electrospray emitter. References 1. Shen, Y.; Zhao, R.; Berger, S.J.; Anderson, G.A.; Rodriguez, N.; Smith, R.D. Anal. Chem. 2002, 74, Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. U.S./Canada (847) Brazil (55) Austria (43) Benelux (31) (32) Denmark (45) France (33) Germany (49) Ireland (353) Italy (39) Sweden (46) Switzerland (41) United Kingdom (44) Australia (61) China (852) India (91) Japan (81) Korea (82) Singapore (65) Taiwan (886) PN _E 07/16S