n a n oA C Q U I T Y U P L C S yst e m w it h 2 D T e c h n o l o g y
A better way to see 2D
T HE DATA YOU NEED TO QUANT IF Y C HANGES IN P ROT EIN E X P RESSION
As protein analyses move from discovery toward clinical applications, reproducibility and robustness become increasingly important:
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Header 2
Labs seek to improve reproducibility in terms of technical replication on the same instrument,
Bullet Sidebar Antitled as well as duplication of results between laboratories.
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To identify proteins at lower concentrations, labs are turning to two-dimensional separations at
Medium 9.5/15 pt
the protein and the peptide level.
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Two-dimensional (2D) chromatography traditionally has been performed with strong cation
exchange (SCX) followed by reversed phase (RP) due to the orthogonal nature of the separation
mechanisms. The major disadvantage of this approach is that, due to the limited resolution capabili-
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idunt praessi
ties provided by SCX, peptides often are split across first-dimension fractions…until now.
The nanoACQUITY UPLC® System with 2D technology adds a new magnitude to orthogonal
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separations by employing two reversed-phase separations at high and low pH: high-resolution
faccum vel essi er autpat
separations are provided in both dimensions, instead of one low-resolution (typically SCX) and one
Ut ipis ex et praesectem high-resolution separation (typically RP). UPLC® is a proven technology that provides significantly
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better reproducibility, larger dynamic range, and dramatically less fraction-to-fraction carryover
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for protein analyses.
3 0.6 8
100
pH 10.0
20 mM ammonium formate,
1 8 .9 5
4 1 .2 6
2 5.68
%
5 .7 0 6.77
9 .6 4
5 .2 1
13 .2 1
1 6.4 1
1 8 .5 8
B in 7 0 m i n u t e s
20 m M
4 1 .9 2
2 9 .0 0
3 9 .5 8
1 0 .0 0
T im e
1 2.5 0
15 .00
1 7 .5 0
2 0 .0 0
2 2 .5 0
neutral acidic
basic
100
pH 2.6
2 5 .0 0
2 7.5 0
30 .00
3 2 .5 0
3 5 .0 0
3 7.50
4 0 .0 0
basic
0.3% formic acid,
1 7 .3 6
26 .5 1
1 6 .3 0
2 2.7 9
3 5 .0 5
3 0 .6 8
1 1 .4 0
1 3.2 4
4 .2 9
1 1 .9 3
1 9.6 1
1 9 .9 3
14 .09
2 6 .0 6
columns help optimize
chromatographic resolution with
BEH Technology™
in 50 min+
3 1.4 1
2 2 .3 9
6 .2 9
nWaters’
0% to 42% acetonitrile
2 7 .0 0
2 6 .6 8
%
4 .70
1 : S 45
c a .0
n 0E S +
acidic
2 3 .8 6
1 0 .9 9
4 2 .5 0
28 .55
1 8.7 5
8 .9 1
n High-pressure fluidic modules
(up to 10,000 psi)
2 4.2 0
N H 4O H pH 1 0
0 in_ D ig e 5.00
B o v ine _ H em 2o .5
glob
s t_S to re d _ 0971.58 00 3 _1
Automated system set-up and
operation
35 .48
2 1.0 4
19 .89
18 .21
0 -5 16 %
2 5.9 2
2 2 .3 9
1 1 .7 3
n
in 50 min
1 4 .0 3
10 .38
4 .10
3 7 .6 5
29 .41
1 5 .7 9
8 .5 3
0% to 42% acetonitrile
3 5 .8 5
nanoACQUIT Y UPLC with
2D technology provides:
3 4.27
3 6 .1 9
n
1
Comparison of peptide elution at pH 10.0 (top) and pH 2.6 (bottom) showing good UPLC
chromatographic resolution and change in elution order.1
Direct nano-flow, without splitting, for low solvent usage
n Superior reproducibility and
reliability for flow rates from
micro-scale to nano-scale
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Outstanding performance built on
proven UPLC technology
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Ease-of-use for every type of lab
and chemists of all skill levels
SU P ERIOR 2D SYST EM T EC HNOLOGY
Trap Column
2
1
4
Trap Valve
4
5
V
Syringe
3
RP Column 1
Nano Tee
BSM2
6
Plug
3 Inject Valve 6
1
5
g
2
Plu
Needle
Analytical Column
Nano Tee
BSM1
Waste
The system’s integrated valves remove the need for manual fraction collection.
Waters’ patented valve technology automates the analytical transfer
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from high to low pH, replacing cumbersome manual fraction collection, extraneous dry-down time, and possible sample loss. This
The first-dimension binary solvent manager (BSM2) elutes
analytes from RP column 1 to a mixing tee.
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The second-dimension binary solvent manager (BSM1) takes
at-column dilution technique dilutes and acidifies organic eluent to
sample from the first dimension and adjusts the pH from 10 to
pH 2, allowing fractions from the first dimension at pH 10 with high
2 while decreasing the organic content to trap the sample on the
organic content to bind and separate on the second dimension.
trapping column.
How nanoACQUIT Y UPLC-2D at-column dilution works:
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The sample is injected on the first-dimension column (RP column
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The second-dimension BSM1 takes over the separation at pH 2
for high-resolution MS analyses.
1) at pH 10 and unwanted solutes are washed to waste.
2D software wizard helps you manage the process
The intuitive nanoACQUITY UPLC-2D Method Generator walks you
through the necessary steps to develop the right separation method
based on your sample complexity. Simply indicate how many fractions you would like to collect from the first-dimension separation
and the gradient conditions you want to use in the
second dimension.
A NE W DIREC T ION IN ORT HOGONAL SE PA RAT IONS
Increased resolution translates into better protein identification
The nanoACQUITY UPLC-2D provides high peak capacity, recovery
While 1D-separations with sub-2 µm particles will provide high
of more peptides, and greater protein coverage,
quality results, nanoACQUITY UPLC-2D resolution allows you to
with greater confidence in results.
obtain an increased number of secure identifications. The Venn diagram (Figure 2), shows the same results depicted in the multi-step
1D
365 Proteins
2874 Peptides
2000
chromatograms (Figure 1), by illustrating overlap and expanded
information with increased steps.
1500
1000
1D
365 Proteins
500
0
20
40
60
80
100
R e te n tio n T im e (m in )
2D – 5 Steps
695 Proteins
7961 Peptides
2D – 10 Steps
778 Proteins
100
%
0
2 0 .0 0
3 0 .0 0
4 0 .0 0
5 0 .0 0
6 0 .0 0
7 0 .0 0
8 0 .0 0
9 0 .0 0
T im e
2D – 5 Steps
695 Proteins
Figure 2. From a 1D analysis of E. coli, 365 proteins were identified in two of three
replicate injections. Almost all of these proteins were identified in the 2D experiment,
where 695 proteins were identified in two of three replicates. The number of peptides
increased dramatically between the 1D experiment (2874 peptides) and the 2D
experiment (7961 peptides).3
2D – 10 Steps
778 Proteins
9415 Peptides
Get the most information from your
smallest samples:
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Analyze irreplaceable samples with biomarkers
expressed in femtomoles and below
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100
%
0
20 .0 0
3 0 .0 0
4 0 .0 0
5 0 .0 0
6 0 .0 0
7 0 .0 0
8 0 .0 0
9 0 .0 0
T im e
Figure 1. In this separation, the power of nanoACQUITY UPLC with 2D for resolving
complex proteomic samples is demonstrated by this tryptic digest of E. coli. Using
nanoACQUITY UPLC in one dimension, 0.5 µg were loaded on-column to resolve and
identify 365 proteins from 2874 peptides. Identification of 7961 and 9415 peptides
from the five-step and 10-step separations, respectively, were obtained by adding
2.5 µg to the system.2
Simplify cellular lysate or plasma samples for better
MS data quality
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Pinpoint changes in glycosylation, phosphorylation,
or other post-translational modifications among less
important proteins
Reproducibility across the hall and
around the world
TOTAL SYST EM SOLUT IONS FOR P ROT EIN ANAYLSIS
With an integrated LC/MS system matched with specialized software
MassLynx™ Software, and powerful application managers, the
for protein expression analysis, your laboratory is in a stronger
nanoACQUITY UPLC System with 2D technology extends nano-scale
position to cross the bridge from discovery to validation workflow
separations analyses for confident identification and best-in-class
for proteomic samples.
reproducibility.
Laboratory 1
690 Proteins
When used with Waters SYNAPT™ and Xevo™ MS technologies,
Laboratory 2
595 Proteins
Common 538
Figure 3. The five-fraction E. coli experiment was repeated in two laboratories, on
different instruments, and by different operators, to test the reproducibility of the
nanoACQUITY UPLC-2D System. In laboratory 1, 690 proteins were identified in two of
three replicate injections, while in laboratory 2, the number was 595 proteins. The Venn
intersection shows that more than 90 percent (538) of these proteins were identified in
both laboratories.4
1. Two-dimensional separation of peptides using RP-RP-HPLC system with
different pH in first and second separation dimensions. Gilar M, Olivova P,
Daly AE, Gebler JC. J Sep Sci. 2005 Sep;28(14):1694-703.
2,3,4. A reproducible online 2D reversed phase-reversed phase high-low pH
method for qualitative and quantitative proteomics. Stapels M, Fadgen K.
Spectroscopy, in press.
Waters, nanoACQUITY UPLC, and UPLC are registered trademarks of
Waters Corporation. BEH Technology, MassLynx, SYNAPT, Xevo, and The
Science of What’s Possible are trademarks of Waters Corporation. All other
trademarks are the property of their respective owners.
©2009 Waters Corporation. Produced in the U.S.A.
March 2009 720002996EN PC-PDF
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