Development and Performance Evaluation of a Robust nano LC-MS/MS Vented Column Set-up with 25 µm ID

Columns for Improved Sensitivity

Mauro De Pra1, Thomas Koecher2; Peter Pichler3; Laurent Rieux1; Remco Swart1; Karl Mechtler2, 4
1 Thermo Fisher Scientific, Amsterdam, NETHERLANDS; 2 IMP, Vienna, AUSTRIA; 3 Christian Doppler Laboratory for Proteome Analysis, Vienna, AUSTRIA;
4 IMBA, Vienna, AUSTRIA

Splitless Nano Pump Performance

Overview 6 FIGURE 4. Comparison of
The UltiMate™ RSLCnano UHPLC system was adapted to meet the requirements the peak width at half height
Purpose: An easier and robust solution to nano LC-ESI-MS at ultra-low flow
of ultra-low nano flow. The flow selector was replaced with a prototype version 5,5 in direct injection and pre-
rates is demonstrated.
suitable for controlling flow rates lower than 70 nL/min. concentration. 30 min
PWHH (sec)
Methods: 25 μm ID columns were operated with a split-less pump at flow rates 5 direct injection gradient from 4 to 55% B at
The flow delivery precision of the pump was tested by evaluating the relative
lower than any existing commercial system. Pre‐concentration 50 nL/min. Sample loading
standard deviation (RSD) of the retention time of the peptide peaks. The precision 4,5
at 2 μL/min. UV detection
Results: Performance of the column and UHPLC system were excellent. was outstanding throughout the flow range used for the experiments (Table 1 and
4 (214 nm).
Preliminary data on MS sensitivity are shown. Table 2).
1 2 3 4
The prototype flow selector can be easily and reversibly implemented in any
UltiMate 3000 RSLCnano system.
Introduction Effect of Flow Rate and Gradient Length
Increased sensitivity in nano LC-ESI-MS is generally achieved by decreasing the TABLE 1. Retention time precision TABLE 2. Retention time precision
As was expected from nanospray theory, lowering the flow rate below 50 nL/min
inner diameter (ID) of the column, resulting in higher peak concentration and more of 6 peptides at 20 nL/min. The rest of a selected peptide at various flow
resulted in an increase in signal intensity (data not shown). This, consequently,
ions detected with mass spectrometry. of the conditions are the same as rates. Conditions are the same as
resulted in an increased number of identified peptides and proteins (Figure 5 and
Figure 1. Figure 1 except for the flow rate.
6). Lowering the flow rate from 50 to 20 nL/min resulted in a 20% increase in both
Consequently, more peptides can be successfully sequenced and thereby
peptide and protein IDs. As previously found2, the use of long gradients also
improve protein identification. The number of peptides detected in complex Peak RT (min) RSD (%) Flow Rate (nL/min) RT (min) RSD (%) resulted in an increase in peptide and protein IDs. This can be explained by the
samples can increase by more than a factor of 100 just by decreasing the column
1 49.67 0.06 20 54.5 0.04 increased peak capacity provided by longer gradients. The relation between
diameter from
increased peak capacity and peptide and protein IDs peptide and protein IDs is
75 µm to 30 µm ID1. 2 51.72 0.04 30 37.67 0.03
shown in Figure 7 and 8.
3 54.5 0.04 40 30.84 0.03
With the reduction in column ID, the gradient flow delivered by the pump needs to
4 54.91 0.03 50 26.22 0.08 FIGURE 5. Number of
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 5 57.36 0.03 identified peptides
was traditionally achieved by implementing a custom-made flow splitter in the 6 59.77 0.03 using a 25 μm ID
commercial systems. column and different
gradient times.
In the present study, we used a split-less pump tailored to the flow requirements of Pre-Concentration with Vented Column Sample: 100 ng HeLa
peptide analysis using 25 µm ID columns. lysate.
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
Methods 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
Liquid Chromatography pressurization during the whole run, irrespective of the valve position.
FIGURE 6. Number of
•Nano UHPLC: Thermo Scientific Dionex UltiMate 3000 RSLCnano system Even when pre-concentration was set up, the precision of the analysis was identified proteins
•Column: 25 µm ID × 15 cm, packed with Thermo Scientific Acclaim PepMap preserved. The flow restrictors were essential to ensure fast and repeatable using a 25 μm ID
RSLC C18, 2 μm, 100 Å pressurization after valve switching. column and different
gradient times
•Trap column: 50 μm ID × 7 cm (2 cm packed bed), packed with Acclaim™ (4–55% B). Sample:
PepMap™ C18, 3 μm, 100 Å 100 ng HeLa lysate.
FIGURE 2. Plumbing schematic for vented column pre-concentration. The
•Mobile Phases: valve can be switched to either direct the flow of the loading pump through
• A: 0.1% Formic acid (FA) in water; the trap column (Top, Loading stage) or to enable the separation of peptides
• B: 0.1% FA in water/ACN (2/8), gradients indicated with figures at low nL/min flow rates (bottom, elution).
• Loading solvent: 0.1% trifluoro acetic acid (TFA) in water at 1.5 μL/min.

Mass Spectrometry FIGURE 7. Relation between chromatographic peak capacity and number of
identified peptides (top) and proteins (bottom) using a 25 μm ID column.
•Thermo Scientific Q Exactive mass spectrometer Sample: 100 ng HeLa lysate.
•New Objective FS-360-20-5-D-5 nano-ESI emitter
•Peptides are ionized via electrospray and analyzed via data dependent MS/MS

Data Analysis
•Thermo Scientific Dionex Chromeleon 6.8 Chromatography Data System
•Thermo Scientific XCalibur 2.2 Data Acquisition System
•Thermo Scientific Proteome Discoverer software; FDR 1%

Results FIGURE 3. Overlay of six consecutive injections with sample preconcentration

Column Performance
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 fitted with
Thermo Scientific Dionex nanoViper fingertight fittings capable of withstanding
UHPLC pressures up to 1000 bar. 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).
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). 4─55% B in 30 minutes at 50 nL/min. The peaks label is the
width at half height.
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). 4─55% B in
60 minutes at 50 nL/min. Sample loading at 2 μL/min. UV detection (214 nm)
Conclusion
ƒ We established a user friendly LC system operable at flow rates of 20 nL/min.
ƒ High performance 25-μm ID columns packed with 2 µm C18 stationary phase
were developed and tested.
ƒ The Ultimate 3000 RSLCnano was configured to efficiently deliver flow
between 20 and 50 nL/min without splitting. Peak widths at flow rates
20 nL/min are comparable to regular nano LCs (operated at ~ 250 nL/min).
ƒ RSD of retention times of less than 0.2% over three months of operation
ƒ Coupling to MS with ESI emitters with 10 µm ID and tip opening 5 µm showed
the expected dependence of electrospray ionization on the flow rate.
ƒ Up to 25,000 peptides were identified in a single nano LC-MS run.

References
1. Shen, Y.; Zhao, R.; Berger, S.J.; Anderson, G.A.; Rodriguez, N.; Smith, R.D.
Anal. Chem. 2002, 74(16), 4235-4249.
2. Kocher, T.; Swart, R.; Mechtler, K. Anal. Chem. 2011, 83(7), 2699-2704.

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 two hydrophilic and two hydrophobic peptides showed that
the peak width increase is well below 1 second.
Preliminary UHPLC-UV-MS Results
Acknowledgements
We would like to thank Johannes Fuchs and Gabriela Krssakova from Karl
Mechtler‘s lab at the Institute of Molecular Pathology in Vienna, Austria.
All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
PO70259_E 08/12S

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.