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[ CARE AND USE MANUAL ]
MassPREP On-Line Desalting Cartridge
CONTENTS
I. INTRODUCTION
II. INSTALLING THE MASSP REP ON-LINE
DESALTING CART RIDGE INTO T HE SENTRY
2.1 X 10 MM GUARD COLUMN HOLDER
III. RECOMMENDED LC/MS SYSTEM CONFIGURATION
TO MINIMIZE MS SOURCE CONTAMINATION
IV. SUGGEST ED METHOD FOR MASS ANALYSIS
USING MASSPREP ON-LINE DESALTING
CARTRIDGE
a. Instrumentation
b. Separation Method
V. GUIDELINES FOR USE AND
TROUBLESHOOTING
VI. MISCELLANEOUS
I. INTRODUCTION
Mass spectrometry is a powerful tool for intact protein analysis.
However, the quality of analyses can be compromised by salts in
the sample that interfere with ionization and/or form adducts with
the investigated proteins. It is therefore important to remove or
significantly minimize the introduction of extraneous salt ions
into the mass analyzer. The use of on-line, desalting devices can
efficiently perform this function with minimal sample handling.
Waters MassPREP™ On-line Desalting Cartridges (2.1 x 10 mm,
P/N 186002785) contain a polymer-based, reversed-phase
packing material that can be used to effectively desalt protein
samples prior to mass spectrometry. (Note: The purchase and use
of Waters Sentry™ 2.1 x 10 mm Guard Column Holder Kit (P/N
WAT097958) is required for this application as shown in Figure 1.)
The phenyl phase material contained in these on-line devices
successfully “traps” proteins allowing the salts to be washed
to waste prior to elution of the desalted protein into the mass
spectrometer. With an optimized LC/MS method, cycle times as low
as 3 minutes are achievable. These on-line devices, when used with
an appropriate HPLC System injector and injector wash solution,
can be used for the sequential analyses of different samples (See
Figures 2 and 3).
This Care and Use manual provides suggested methods for the
successfully use of this product. For the latest list of MassPREP,
BioSuite™ or NanoEase™ column offerings, go to www.waters.com/
lifesciences.
II. INSTALLING THE MASSPREP ON-LINE
DESALTING CART RIDGE INTO T HE SENTRY
2.1 X 10 MM GUARD COLUMN HOLDER
1. Unscrew the two halves of the Sentry 2.1 x 10 mm Guard
Column Holder.
2. Remove the old MassPREP On-line Desalting Cartridge, if
present.
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3. Install the new MassPREP On-line Desalting Cartridge, noting
the flow arrow direction on the device. This should match the
flow arrow direction on the analytical column.
4. Reassemble the Sentry 2.1 x 10 mm Guard Column Holder.
Hand-tighten only.
5. Connect the 2.1 x 10 mm Guard Column Holder containing
the MassPREP On-line Desalting Cartridge onto HPLC or
LC/MS System using appropriate connectors (e.g., PEEK™
tubing and connectors). Prior to use, the MassPREP On-line
Desalting Cartridge reversed-phase packing material must
be “wet” with organic solvent prior to equilibration with 95%
Eluent A/5% Eluent B. This can be accomplished by running
a “blank injection” (i.e., buffer injection followed by running
the separations gradient) or by washing the column with a
minimum of five column volumes of 50% Eluent B followed by
a ten column volume equilibration at 95% Eluent A/5% Eluent
B prior to sample injection. While equilibrating, check the
holder and all connections for leaks.
III. RECOMMENDED LC/MS SYSTEM
CONFIGURATION TO MINIMIZE MS SOURCE
CONTAMINATION
The presence of non-volatile salts (e.g., NaCl) can suppress protein
ionization and foul the ionization source of the mass spectometer.
It is therefore important to remove or significantly minimize the
introduction of these compounds into the mass analyzer. Figure
2 details an LC/MS configuration that can effectively be used to
load sample onto the column and elute salt to waste prior to valve
switching and protein elution into the mass analyzer.
Position 1: Sample Loading and Salt Elution to Waste
Spectrometer
MassPREP
On-Line
Desalting
Cartridge
Waste
1
2
3
6
5
4
Gradient
HPLC
Mass
Figure 1: Sentry Guard Holder (P/N WAT097958) and MassPREP On-line
Desalting Cartridge (P/N 186002785).
HPLC
Pump
Recirculation
Position 2: Sample Elution to Mass Spec
Spectrometer
MassPREP
On-Line
Desalting
Cartridge
HPLC
Pump
Waste
1
2
6
5
4
3
Recirculation
Gradient
HPLC
Mass
Figure 2: LC/MS Valve Switching Configuration for use with MassPREP
On-line Desalting Cartridge.
MassPREP On-Line Desalting Cartridge
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IV. SUGGEST ED METHOD FOR MASS ANALYSIS
USING MASSPREP ON-LINE DESALTING CART RIDGE
(Considerations to Minimize Protein Carryover
Between Injections):
HPLC injector design, composition of injector wash solution, and
number of injector wash cycles between injections are some of the
factors that can effectively minimize protein carryover between
sample analyses. Waters MassPREP On-line Desalting Cartridges,
when used with an appropriately performing HPLC System and
injector wash protocol, can be used for the sequential analyses of
different samples due to the lack of protein carryover from prior
injections. Figure 3 shows the mass analyses results obtained
from buffer blank and intact protein injections [bovine serum
albumin (BSA) and IgG monoclonal antibody (mAb)] processed
through the MassPREP On-line Desalting Cartridge using the
Waters instrumentation and separation method detailed below:
a. Instrumentation
HPLC System: Alliance® 2796 Separations Module
Needle Wash
Solution Composition: H20 (35%) / Isopropanol (5%)
Acetonitrile (60%)
Number of Needle Washes: Single
Needle Wash Time: 15 sec needle exterior/3 sec inject port
b. Separation method:
Eluent A: H20 with 0.1% Formic Acid
Eluent B: Acetonitrile with 0.1% Formic Acid
Flow: 0.4 mL/minute
Initial Column Conditioning:
Prior to use, the MassPREP On-line Desalting Cartridge reversed
phase packing material must be “wet” with organic solvent prior
to equilibration with 95% Eluent A/5% Eluent B. This can be
accomplished by running a “blank injection” (i.e., buffer injection
followed by running the separations gradient) or by washing the
cartridge with a minimum of five column volumes of 50% Eluent
B followed by a ten cartridge volume equilibration at 95% Eluent
A/5% Eluent B prior to sample injection.
Sample Loading/Salt Elution:
Load sample onto cartridge while delivering 95% Eluent A/5%
Eluent B. The protein sample will bind to the cartridge while the
contaminating salt passes through unretained to waste.
Sample Elution to Mass Spec:
At approximatly. 0.5 min, begin increase Eluent B concentration
from 5 to 80% in 1.5 min. Hold at 80% B for an additional 1.0 min
to allow protein to completely elute from cartridge into MS system.
Mass Spectrometer: Q-tof
Ionization mode: ES +
Capillary voltage: 3300
Cone voltages: 30 for BSA mass analysis 35 for
mAb mass analysis
Source temp: 150 °C
Desolvation temp: 350 °C
Gas flow: 500 L/Hr
Cone gas: 50 L/Hr
Column re-equilibration:
At approximately 3.0 min, re-equilibration cartridge with ten
cartridge volumes of 95% Eluent A/5% Eluent B prior to next
sample injection.
MassPREP On-Line Desalting Cartridge
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Injection 100_blank
100
%
0
Injection 99_MAb
100
%
0
100
Injection
%
0
100
Injection
%
0
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
Time
100
%
0
100
%
0
100
%
0
100
%
Injection 100 blank
Injection 99 MAb
Injection 98 BSA
Injection 97 blank
0
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800
m/z
Figure 3: Results from final 4 injections on MassPREP On-line Desalting Cartridge from a 100 injection overnight sequence noting absence of protein
carryover between injections. Test series consisted of repetitive injections of PBS, IgG monoclonal antibody (mAb) and BSA.
MassPREP On-Line Desalting Cartridge
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V. GUIDELINES FOR USE AND T ROUBLESHOOTING
1. The amount of intact protein to load onto a MassPREP On-line Desalting Cartridge to obtain
acceptable mass analysis results depends upon a number of variables. These include the
type of mass analyzer used, the ionization efficiency of analyzed protein and MS source, the
potential ionization suppression effect of ion-pairing reagents used in the separation eluent
(e.g., TFA), etc. In general, between 1 and 5 µg of injected protein such as BSA or an IgG
monoclonal antibody should yield good mass detector signal and excellent protein recovery
from the MassPREP On-line Desalting Cartridge. Proportionately less material should be
injected when analyzing smaller molecular weight proteins to prevent overloading the On-line
Desalting Cartridge and or mass detector.
2. The maximum recommended operating pressure for MassPREP On-Line Desalting Cartridge,
when configured WITHOUT pre-column tubing, is approximately 150 psi. Use of pressure
generating tubing immediately before the MassPREP On-Line Desalting Cartridge is required
to generate sufficient UPLC System back pressure for proper functioning. Thus, the total
reported UPLC System backpressure generated when BOTH the pre-column tubing and
MassPREP On-Line Desalting Cartridge are configured for an application is approximately
5700 psi at a flow of 0.5 mL/min with a 5% ACN/0.1% FA eluent.
3. Results obtained with Waters MassPREP On-line Desalting Cartridges can vary depending
upon the physical characteristics of the analyzed proteins as well as on the performance
characteristics of the HPLC (or LC/MS System), injector, and injector wash protocol used.
VI. MISCELLANEOUS
Recommended Storage:
For overnight storage, continuously flush the cartridge with the mobile phase at slow flow (e.g.,
0.1 – 0.2 mL/min). If the cartridge is not to be used for several days, store the cartridge in 50%
acetonitrile (CH3CN) without any eluent additives such as formic acid or TFA. Do not store the
MassPREP On-line Desalting Cartridges in buffered eluents. Completely seal the stored column to
avoid evaporation and drying of the bed.
Waters Corporation, Waters. The Science of W hat’s Possible, and Alliance are registered trademarks of Waters Corporation. MassPRE P, Sentry,
BioSuite, NanoEase, and Q-Tof micro are trademarks of Waters Corporation. All ot her trademarks are property of their respective owners.
©2014 Waters Corporation. Produced in the U.S.A. June 2014 715001026EN Rev C. KP-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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