Waters MassPREP Micro Desalting Column User Manual

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MassPREP Micro Desalting Column

CONTENTS

I. INTRODUCTION

II. CONNEC TING THE MASSPREP MICRO
DESALTING COLUMN WITH T HE CHOSEN
LC SYSTEM

III. LC/MS SYSTEM CONFIGURATION TO
MINIMIZE MS SOURCE CONTAMINATION

IV. USING MASSP REP MICRO DESALTING
COLUMN TO MINIMIZE PROTEIN CARRYOVER
BETWEEN INJECTIONS

V. GUIDELINES FOR USE

VI. RECOMMENDED STORAGE

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 spectrometer. The use of on-line, desalting devices can
efficiently perform this function with minimal sample handling.

Waters MassPREP™ Micro Desalting Columns (2.1 x 5 mm, P/N

186004032) contain a polymer-based, reversed-phase packing
material (20 µm particle size, 1000 Å pore size) that can be used
to effectively desalt protein samples prior to mass spectrometry.
The phenyl phase material contained in these on-line devices
successfully retains 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 4 minutes are achievable. These on-line devices, when used with
an appropriate LC system injector and injector wash solution, can
be used for the sequential analyses of different samples.

II. CONNECTING THE MASSP REP MICRO DESALTING
COLUMN WITH T HE CHOSEN LC SYSTEM

The MassPREP Micro Desalting Column can be used with an
appropriately performing LC system to perform the desalting
functions so long as proper fittings/tubings are used to make
connections. However, optimized performances are achieved
only when the cartridge is connected to a Waters ACQUITY or
nanoACQUITY UPLC system. For the use of the cartridge with
ACQUITY or nanoACQUITY, pre-assembled tubing kits (Intact Mass
Tubing Kit, p/n 205000548) are provided to connect the column to
the UPLC systems and mass spectrometer.

There are two configurations to connect the cartridge into the LC
fluidic path. The inlet of the cartridge can be directly screwed onto
the Valco® UPLC injection valve with no tubing connection.

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This configuration would leave the cartridge operating at room
temperature. The other configuration is to screw the cartridge
into one end of the included union connector. The connection of
proper tubing to the other end of the union connector would allow
the cartridge to be placed into a column heater to control the
desalting temperature.

III. LC/MS SYST EM CONFIGURATION TO
MINIMIZE MS SOURCE CONTAMINATION

Non-volatile salts (e.g., NaCl) can suppress protein ionization
and foul the ionization source of the mass spectrometer. It
is, therefore, important to avoid the introduction of these
compounds into the mass spectrometer. Figure 1 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 spectrometer.

Position 1: Sample Loading and Salt Elution to Waste

MassPREP

Gradient
UPLC/HPLC
System

Micro
Desalting
Column

Mass
Spectrometer

Position 2: Sample Elution to Mass Spectrometer

MassPREP

Gradient
UPLC/HPLC
System

Figure 1. LC/MS Valve Switching Configuration for Use of MassPREP
Micro Desalting.

Micro
Desalting
Column

Mass
Spectrometer

Waste

Waste

IV. USING MASSPREP MICRO DESALTING
COLUMN TO MINIMIZE PROTEIN CARRYOVER
BETWEEN INJECTIONS

LC 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 Micro Desalting Column, when
used with an appropriately performing UPLC/HPLC system and
injector wash protocol, can be used for the sequential analyses
of different samples due to the removal of protein carryover from
prior injections.

V. GUIDELINES FOR USE

1. Initial Column Conditioning. Prior to use, the MassPREP Micro
Desalting Column reversed-phase packing material must be
“wet” with organic solvent prior to equilibration with 95%
Eluent A / 5% Eluent B (e.g., Eluent A = 0.1% formic acid in
MS grade H20 and Eluent B = 100% Acetonitrile). This can
be accomplished by running a “blank injection” (i.e., Eluent A
injection followed by running the separation gradient) or by
washing the column with a minimum of five column volumes
(~20 µL) of 50% Eluent B followed by a ten column volume
equilibration at 95% Eluent A / 5% Eluent B prior to sample
injection.

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 Micro Desalting
Columns can vary depending upon the physical characteristics
of the analyzed proteins as well as on the performance
characteristics of the HPLC (or UPLC/MS System), injector, and
injector wash protocol used.

MassPREP Micro Desalting Column

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