Protea Biosciences, Inc.
955 Hartman Run Road
Morgantown, WV 26507
P: 304.292.2226
F: 304.292.7101
www.proteabio.com
ProgentaTM Zwitterionic Acid Labile Surfactants
ALS-200-5mg / ALS-200-5x5mg / ALS-200-10x5mg
ALS-210-5mg / ALS-210-5x5mg / ALS-210-10x5mg
I.
Product Description
Progenta Zwitterionic Acid Labile Surfactants (ZALS) are novel, acid cleavable
detergents that are fully compatible with mass spectrometry analysis. The
Progenta ZALS’s have been engineered to provide a safe alternative to
detergents (e.g. CHAPS) that are commonly used in proteomics work, but that
negatively impact subsequent analysis by mass spectrometry. While CHAPS
and other detergents can improve protein solubility, they can be very difficult to
remove during sample prep and purification of the protein sample. These bound
detergents can cause significant impairment of protein analysis by mass
spectrometry, as the surfactants can suppress analyte ion signal, promote
analyte adduct formation, and present as contaminants during the analysis.
The Progenta Zwitterionic Acid Labile Surfactants are fully mass spec compatible
and alleviate the problems commonly associated with CHAPS and other
detergents in proteomics studies. At neutral pH, the Progenta ZALS functions as
a powerful detergent for use in sample preparation, protein solubilization, and cell
lysis protocols. After completing the experimental work, the solution is adjusted
to a pH of 2.5 with trifluoroacetic acid (TFA) and incubated for 30 minutes to fully
cleave the ZALS into small organic molecules that do not exhibit surfactant
activity or interfere with analysis by mass spectrometry. Other commercially
available acid labile surfactants require incubation in harsher acidic environments
of pH 1.0 to 2.0 for up to several hours and may produce an oily pellet or film
from the cleavage by-products. Progenta ZALS has been specially engineered
to provide powerful surfactant performance with the added benefit of quick,
simple acid-based detergent cleavage and removal for reproducible results in
mass spectrometry-based proteomics studies.
Both Progenta Zwitterionic Acid Labile Surfactants are effective at solubilizing
peptides and proteins, functioning in cell lysis protocols, optimizing enzymatic
digestions, reducing surface adsorption losses via non-specific interactions, and
replacing CHAPS in the isoelectric focusing (IEF) step of 2D gel separations.
ZALS I has a Critical Micelle Concentration (CMC) of 3.4 mM. ZALS II has a
shorter hydrophobic tail, yielding a less powerful CMC of 31.3 mM. Both ZALS I
and II exhibit fast, efficient degradation kinetics at acidic pH’s of 2.0 to 2.5.
II.
Background Information
Not applicable
Protea Biosciences, Inc.
955 Hartman Run Road
Morgantown, WV 26507
P: 304.292.2226
F: 304.292.7101
www.proteabio.com
III.
Research Applications
 Solubilization of proteins and other biomolecules
 Sample preparation and solid phase extraction
 Improved enzymatic digestion of proteins
 Cell lysis and protein extraction from cell lines, tissues, and biological
samples
 2D gel electrophoresis buffers
 Extraction of biomolecules from environmental samples
IV.
Features and Benefits
 Rapidly cleaved in just 30 minutes at pH 2.0 – 2.5 in 1% TFA
 ZALS cleavage products are fully soluble and can be washed away to not
interfere with mass spectrometry analysis
 Promotes protein solubility and improves enzymatic digestion
 Certified Mass Spec Grade
V.
Instructions for Use (Technical Manual content)
Reconstitution of Zwitterionic Acid Labile Surfactant (ZALS)
The ZALS working stock can be made up in several different solvents for
subsequent usage. Recommended solvents include water, 0-50% methanol, 050% acetonitrile, 5-50 mM ammonium bicarbonate buffer, 5-50 mM Tris-HCl
buffer, 5-50 mM sodium phosphate buffer, and 5-50 mM ammonium acetate
buffer. It is important that the solvent pH be between 7 and 10 to prevent
degradation of the surfactant, so the pH of each solvent should be checked prior
to usage.
Table 1 shows the volumes required to reconstitute 1 mg and 5 mg vials of the
ZALS powder. The recommended working concentration for using the ZALS is
0.01 to 0.1%. Surfactant cleavage will occur within 30 minutes in a 1% TFA
solution (water-based solvent).
Table 1: Reconstitution of Zwitterionic Acid Labile Surfactant (ZALS) Powder
1 mg ZALS Powder
5 mg ZALS Powder
Volume of Buffer to
Add to ZALS Powder
ZALS
Concentration (w/v
in g/mL)
Volume of Buffer to
Add to ZALS Powder
ZALS
Concentration (w/v
in g/mL)
1.0 mL
0.1%
5.0 mL
0.1%
500 µL
0.2%
2.5 mL
0.2%
200 µL
0.5%
1.0 mL
0.5%
100 µL
1.0%
0.5 mL
1.0%
Protea Biosciences, Inc.
955 Hartman Run Road
Morgantown, WV 26507
P: 304.292.2226
F: 304.292.7101
www.proteabio.com
General Usage Considerations
The Progenta Zwitterionic Acid Labile Surfactant functions as a CHAPS analog
and it can be used in any experiment where CHAPS is routinely used. For most
methodologies, we recommend a working concentration of 0.01-0.1% ZALS. In a
solution phase experiment, 1.0% trifluoroacetic acid (TFA), pH 2.0 – 2.5 will
efficiently cleave the ZALS detergent within 30 minutes to produce degradation
by-products that do not possess surfactant properties, do not bind to the sample
or reversed phase media, or interfere with mass spectrometry analysis. Other
acids can be used to degrade the ZALS detergents, but their effects on
subsequent sample preparation and analysis must be considered carefully.
Protocol for Protein Digestion Using the Progenta Zwitterionic Acid Labile
Surfactant
Materials Needed:






Progenta Zwitterionic Acid Labile Surfactant (Cat. No. #ALS-200-5mg or
#ALS-210-5mg)
50 mM ammonium bicarbonate buffer, pH 7.8
500 mM dithiothreitol (DTT)
500 mM iodoacetamide (IA)
10% trifluoroacetic acid (TFA)
trypsin, mass spectrometry grade (250 ng/µL in 0.01% acetic acid)
Digestion Protocol for MALDI TOF/TOF:










Make a 0.01% ZALS solution in 50mM ammonium bicarbonate, pH 7.8
(digestion buffer).
Reconstitute the protein sample directly in the 0.01% ZALS digestion solution
in a microcentrifuge tube.
o If the protein sample is already in a solution format, then a 0.02%
ZALS solution in 50 mM ammonium bicarbonate can be made and
used to dilute the protein sample directly in a 1:1 ratio.
o If the protein sample is in a pellet form and difficult to dissolve,
additional digestion buffer can be used to help solubilize the sample.
o For difficult to digest protein samples, a higher concentration (e.g.
0.1%) of ZALS can be used.
Vortex or sonicate the sample for 10 seconds.
Add DTT to a final concentration of 10 mM.
Incubate the sample at 55°C for 30 minutes.
Cool the sample to room temperature for 30 minutes.
Add IA to a final concentration of 50 mM.
Incubate the sample at room temperature in the dark for 30 minutes.
Add trypsin for a final concentration of 1:50 (enzyme:protein).
o CaCl2 may be added to a final concentration of 1 mM to improve
tryptic digestion.
Incubate at 37°C for 4-8 hours.
o Proteolytically resistant proteins may require overnight digestion.
Protea Biosciences, Inc.
955 Hartman Run Road
Morgantown, WV 26507
P: 304.292.2226
F: 304.292.7101
www.proteabio.com
Protocol for Hydrolytic Cleavage of the Progenta Zwitterionic Acid Labile
Surfactant





Prior to analysis by mass spectrometry, add trifluoroacetic acid (TFA) to a
final concentration of 1%. (e.g. Add 10 µL of 10% TFA to 90 µL of enzymatic
digestion).
o The cleavage by-products of the ZALS detergent produce a small
sulfate salt. To prevent signal suppression in the mass spectrometer,
the peptide sample should be desalted either by use of a functional
pipette tip (e.g. C18 ProteaTip) or during standard desalting on a
column in an LC-MS experiment.
Allow the surfactant cleavage to proceed for 30 minutes at room temperature.
If buffered solutions are used (e.g. ammonium bicarbonate), they may require
a slightly longer time for surfactant cleavage if the final pH is >3. Additional
TFA can be added to bring the pH to 2.5 – 3.0 to improve the cleavage
kinetics of the hydrolysis reaction.
Elevating the reaction temperature (20-50°C) will improve the cleavage
kinetics.
Lowered reaction temperatures (4-20°C) can be used, but the slower
cleavage kinetics may require longer reaction times (30-60 minutes).
VI.
Product Care and Storage
The Progenta Zwitterionic Acid Labile Surfactants are provided in convenient,
lyophilized 5 mg quantities. When stored unopened at -20°C, the ZALS
detergents have a shelf life of 1 year. The ZALS are hygroscopic and packaged
in an argon atmosphere to prevent water absorption and subsequent
degradation. At neutral pH, the ZALS will slowly hydrolyze (<1% per month).
Under increasingly acidic conditions, as well as with elevated temperatures, the
ZALS’s will hydrolyze more quickly. The ZALS stock solution should be made in
a non-aprotic, neutral or slightly basic pH solvent (pH 7 to 10) to prevent
degradation. Any unused ZALS solution should be stored at 4°C and used within
1 week of reconstitution.
VII.
Specifications
Progenta
Surfactant
Catalog Numbers
Critical Micellular
Concentration
(CMC)
Recommended
Concentrations
ZALS I
ALS-200-5mg
3.4 mM
0.01 – 2.0%
ZALS II
ALS-210-5mg
31.3 mM
0.01 – 2.0%
Protea Biosciences, Inc.
955 Hartman Run Road
Morgantown, WV 26507
P: 304.292.2226
F: 304.292.7101
www.proteabio.com
VIII.
Performance Data
16976
% Intensity
100
0
8479
4.6E+4
16702
8348
5000
10000
15000 20000
Mass (m/z)
25000
30000
A
% Intensity
1165.8
17063
% Intensity
100
100 5175
0
0
5000
10000 15000 20000 25000 30000
Mass (m/z)
16975
8483
8352
16711
12238
5000
10000
15000 20000
Mass (m/z)
B
1307.0
100
% Intensity
% Intensity
25000
30000
C
100
0
5000
3158.6
8482
16971
16702
8484
8355
16974
4896.6
16714
0
10000
15000
20000
Mass (m/z)
D
25000
30000
5000
10000
15000 20000
Mass (m/z)
25000
30000
E
Mass spectra of 20 pmol myoglobin samples that were C8 spotted onto a MALDI plate
pre-spotted with CHCA MALDI matrix.
Key:
A: Control – 20 pmol myoglobin in water
B: Myoglobin in 0.1% Progenta Zwitterionic Acid Labile Surfactant I
C: Myoglobin in 0.1% Progenta AALS I with a 30 minute incubation in 1% trifluoroacetic
acid (TFA), pH 2.5
D: Myoglobin in 0.1% Progenta Zwitterionic Acid Labile Surfactant II
E: Myoglobin in 0.1% Progenta AALS II with a 30 minute incubation in 1% trifluoroacetic
acid (TFA), pH 2.5
Protea Biosciences, Inc.
955 Hartman Run Road
Morgantown, WV 26507
P: 304.292.2226
F: 304.292.7101
www.proteabio.com
100 5183
100 5174
1509.0
% Intensity
% Intensity
1406.7
0
0
5000
10000 15000 20000 25000 30000
Mass (m/z)
5000 10000 15000 20000 25000 30000
Mass (m/z)
A
0
1290.7
16973
8365
16715
5000
10000 15000 20000 25000 30000
Mass (m/z)
C
100
16981
1.1E+4
8486
% Intensity
% Intensity
100 5164 8485
B
16719
8357
0
5000
10000
15000 20000
Mass (m/z)
25000
30000
D
Mass spectra of 20 pmol samples myoglobin that were loaded onto a C4 ProteaTip for
desalting and concentration prior to spotting on a MALDI target (pre-spotted with CHCA
matrix).
A: 0.1% CHAPS
B: 0.1% CHAPS acidified (incubated in trifluoroacetic acid for 30 minutes)
C: 0.1% Progenta Zwitterionic Acid Labile Surfactant I
D: 0.1% Progenta Zwitterionic Acid Labile Surfactant I (incubated in trifluoroacetic acid
for 30 minutes)
Protea Biosciences, Inc.
955 Hartman Run Road
Morgantown, WV 26507
P: 304.292.2226
F: 304.292.7101
www.proteabio.com
Ordering Information (Catalog Grid)
Catalog
Number
Description
Unit
Price per
Unit
ALS-200-5mg
Progenta Zwitterionic Acid Labile Surfactant I
5mg vial
ea
$175
ALS-200-5x5mg
Progenta Zwitterionic Acid Labile Surfactant I
5 x 5mg vial
5 x 5mg
$775
ALS-200-10x5mg
Progenta Zwitterionic Acid Labile Surfactant I
10 x 5mg vial
10 x 5mg
$1400
ALS-210-5mg
Progenta Zwitterionic Acid Labile Surfactant II
5mg vial
ea
$145
ALS-210-5x5mg
Progenta Zwitterionic Acid Labile Surfactant II
5 x 5mg vial
5 x 5mg
$645
ALS-210-10x5mg
Progenta Zwitterionic Acid Labile Surfactant II
10 x 5mg vial
10 x 5mg
$1145
IX.
Photos
Not available
X.
Additional Data (CAN be used for catalog or website)
XI.
Additional Data (NOT FOR CATALOG OR WEBSITE)