Keywords:
LC-MS, nanoElute,
nanoflow, peak area
reproducibility,
retention time
stability, peak width,
peak capacity
Bruker’s nanoElute UHPLC shows
excellent performance and ease of use
for proteomics
Authors: Thomas Kosinski 1, Matt Willets 2, Pierre-Olivier Schmit 3, Gary Kruppa 2
1
Bruker Daltonik GmbH, Bremen, Germany; 2 Bruker Daltonics Inc., Billerica, MA, USA; 3 BrukerDaltonique S.A., Wissembourg, France
Abstract
Bruker’s new nanoElute nanoflow
UHPLC offers excellent chromato-
graphic performance in terms
of separation and reproducibility
with different gradient lengths,
and with or without a trap column.
Introduction
Samples in shotgun proteom-
ics are often highly complex and
can easily contain thousands of
peptides in a narrow mass range
which leads to analytical chal-
lenges. Therefore peptide sepa-
ration is commonly performed by
nano-flow UHPLC, which brings
several advantages, including high
peak capacity and ESI compatible
solvent composition and flow rate.
Despite the increasing speed and
sensitivity of mass spectrometers,
enhancing their ability to analyze
more ions eluting from the column
at any given time, an improvement
of chromatographic separation
still provides a tremendous boost
in the number of identified proteins.
Furthermore, highly reproducible peak
areas and retention times are crucial
for more sophisticated quantitative
analyses. To cover all these demands,
a reliable HPLC system is essential.
In this study we evaluated the four
essential chromatographic perfor-
mance indicators of the Bruker nano-
Elute: retention time variation, peak
area variation, peak width and peak
capacity, to test the nanoElute for the
challenging requirements of sophisti-
cated proteomic analytics.
Experimental
Samples
Aliquots of a commercially available
tryptic digest of HeLa cells were
diluted with 0.1 % formic acid in water
to a concentration of 100 ng/µL.
For chromatographic separation a
curved gradient with three different
lengths was used, as shown in the
table below. The gradient consists of
solvent A, 0.1 % formic acid in water
and solvent B, 0.1 % formic acid in
acetonitrile with the temperature of
the separation column maintained at
50 °C and a flow rate of 400 nL/min.
Four technical replicates were made
with the 90 min gradient and five tech-
nical replicates with the 120 min gra-
dient and the 240 min gradient, each
measured using a setup without a
trap column, and with a trap column.
Switching between setups with and
without a trap column is conveniently
and easily done in the software.
For MS detection an Impact II QTOF
mass spectrometer equipped with
a CaptiveSpray nanoBooster Source
with pure ACN was used. The instru-
ment was operated in ESI positive
mode acquiring full scan MS and MS/
MS data using the InstantExpertise™
routine. This is a self-adapting auto
MS/MS method which is designed
to obtain highest quality results inde-
pendently of the complexity and con-
centration of the sample. It uses an
advanced parent ion selection proce-
dure combined with a variable MS/MS
acquisition rate [1] and is pre-installed
in the impact II instrument control
software (otofControl).
Data Analysis
For analyzing the chromatographic
data Bruker’s Compass DataAnalysis
was used. Eight peptides, which were
evenly distributed in time through the
gradient, were selected from the HeLa
chromatogram. EICs were made for
these peptide masses and the peak
Mass Spectrometry
Instrument:
Bruker impact II QTOF
mass spectrometer
Dry Gas:
3.0 L/min
Ion source:
CaptiveSpray nanoBooster in
positive ion mode
Dry Temperature:
150 °C
Capillary:
1600 V
nanoBooster:
0.20 Bar
Liquid chromatography
Instrument:
Bruker nanoElute™
Gradient
Conditions:
90 min
Gradient
120 min
Gradient
240 min
Gradient
Composition
B
Column:
Acclaim PepMap™ RSLC;
75 µm x 50 cm
0 min
0 min
0 min
2 % B
Mobile phase A: Water, 0.1 % formic acid
60 min
90 min
180 min
15 % B
Mobile phase B: ACN, 0.1 % formic acid
90 min
120 min
240 min
25 % B
Trap column
loading:
100 % mobile phase A
100 min
130 min
250 min
35 % B
Flow rate:
400 nL/min
110 min
140 min
260 min
95 % B
Injection
volume:
2 µL
120 min
150 min
270 min
95 % B
Column oven:
50 °C
area, retention time and peak widths
were compared between the technical
replicates.
Results and Discussion
The gradient length in shotgun pro-
teomics typically varies between
90 min to 240 min. With the pressure
limit of the new nanoElute of 1000 bar,
combined with a column oven, 50 cm
column lengths are possible. The
nanoElute Method Editor allows any
gradient to be generated with only a
few mouse clicks and all typical wash
and equilibration steps are added auto-
matically.
Performance
Highly reproducible chromatographic
separation is essential in any quanti-
tative analysis. The LC must provide
an eluent flow, the gradient mixture
and the injection volume precisely
to ensure the reproducibility of each
chromatographic run. To evaluate the
instrument performance, we have
compared multiple technical replicates
with a 90 min, 120 min and 240 min
gradient and determined the most
important performance criteria.
As is shown in Figure 2, no matter
which setup was used, base peak chro-
matograms were consistent across all
gradient lengths with a time shift only
due to the higher dead volume when a
trap column was used.
High retention time stability and peak
area reproducibility were achieved
with both column configurations
(Figure 3). Retention time drifts of
only a few seconds were obtained
even with the long 240 min gradient.
With all gradient lengths the retention
time variation was less than 0.5 %.
Also the peak area reproducibility was
extremely good with variations of less
than 10 % for most of the comparisons
and ≤5 % for all experiments using a
trap column (Table 1).
As expected the 90 minute gradient
produced the narrowest peaks for
both column setups, 11 s full width
half maximum (FWHM) (Figure 4).
The FWHM values increased propor-
tionally with the gradients length and
resulted in a peak capacity between
500 and 700 (Figure 5). This ensures
the best separation to enable the mass
spectrometer to analyze as many pre-
cursors as possible.
Fig. 1: Bruker nanoElute Method Editor
Fig. 2: Representative Base Peak Chromatograms for all gradient lengths and column setups. Chromatograms in the upper row are obtained by using a trap column
and in the lower row without a trap column.
0
2
4
6
0
2
4
6
8
20
40
60
80
100
0
Time [s]
Intens. x10
7
0
0.5
1.0
1.5
2.0
0
0.5
1.0
2.0
1.5
2.5
20
40
60
80
100
120
140
0
Time [s]
Intens. x10
7
0
0.5
1.0
1.5
0
0.5
1.0
1.5
25
50
75
100
125
150
175
200
225
250
0
Time [s]
90 min Gradient
120 min Gradient
240 min Gradient
Intens. x10
7
w/o trap
w/ trap
w/o trap
w/ trap
w/o trap
w/ trap
Fig. 3: Extracted Ion Chromatograms showing retention time reproducibility of selected peptides across 5 technical replicates using a 120 min gradient.
0
2
4
6
8
9
9
9 9
10
10
10 10
10
11
11 11
13 13
13
12
12
12
12
12
6800
6850
6900
6950
7000
Time [s]
7050
7100
7150
7200
7250
Intens. x10
6
90 min
120 min
240 min
0
5
10
15
20
25
30
Median Peak FWHM (seconds)
w/o trap
w/ trap
90 min
120 min
240 min
0
100
200
300
400
500
600
700
800
Observed Peak Capacity
w/o trap
w/ trap
Fig. 5: Peak Capacity of 12 peptides across each gradient
Fig. 4: Median Peak FWHM of 8 peptides across each gradient
Conclusion
The nanoElute is the perfect companion to the impact II to perform high performance proteomics experiments. It
outperforms state of the art nano-UHPLC systems in terms of performance and reliability combination. At the same
time the nanoElute offers easy to use method configuration and sophisticated diagnostic procedures with a push of
a button.
It allows for:
• High retention time stability
• High Peak Area reproducibility
• Narrow peaks
• High peak capacity
• Simple gradient configuration
• Automatic diagnostic tool
• Trap column switchable with
a push of a button
Median Retention Time Variation
90 min
120 min
240 min
w/o trap
w/ trap
w/o trap
w/ trap
w/o trap
w/ trap
0.16%
0.05%
0.18%
0.35%
0.11%
0.26%
Median Area Variation
90 min
120 min
240 min
w/o trap
w/ trap
w/o trap
w/ trap
w/o trap
w/ trap
13%
4%
8%
5%
3%
4%
Median Peak FWHM (seconds)
90 min
120 min
240 min
w/o trap
w/ trap
w/o trap
w/ trap
w/o trap
w/ trap
11
11
15
16
26
28
Observed Peak Capacity
90 min
120 min
240 min
w/o trap
w/ trap
w/o trap
w/ trap
w/o trap
w/ trap
514
546
577
676
581
620
Table 1: Summery of chromatographic performance values
For research use only. Not for use in diagnostic procedures.
References
[1] Bruker App Note LC-MS 81
Introducing New Proteomics Acquisition Strategies with the compact™ –
Towards the Universal Proteomics Acquisition Method
[email protected] - www.bruker.com
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