INSTRUCTIONS
Pierce High pH Reversed-Phase Peptide
Fractionation Kit
Pub. No. MAN0015701
Pub. Part No. 2162566.1
Rev A.0
84868
Number
Description
84868
Pierce High pH Reversed-Phase Peptide Fractionation Kit, sufficient materials for fractionation of
up to 12 proteolytically digested protein samples before LC/MS analysis
Kit Contents:
Reversed-Phase Fractionation Spin Columns, 12 columns containing 20mg of resin in a
1:1 water/DMSO slurry
Triethlyamine (0.1% in water), 100mL
Storage: Upon receipt store at 4°C. Reagents are shipped at room temperature.
Note: Upon receipt, the spin column resin can have a non-uniform appearance, but this will not affect
performance. To avoid accidental loss of resin material, do not remove the top screw cap until Step 3
of the Conditioning of the Spin Columns procedure.
Table of Contents
Introduction ................................................................................................................................................................................. 1
Procedure Summary..................................................................................................................................................................... 2
Important Product Information .................................................................................................................................................... 2
Additional Materials Required ..................................................................................................................................................... 2
Material Preparation .................................................................................................................................................................... 3
Fractionation of Proteolytic Digests ............................................................................................................................................ 3
Troubleshooting ........................................................................................................................................................................... 4
Related Thermo Scientific Products ............................................................................................................................................ 5
General References ...................................................................................................................................................................... 5
Introduction
The Thermo Scientific™ Pierce™ High pH Reversed-Phase Peptide Fractionation Kit provides an optimized fractionation
protocol and reagents to increase the number of proteins identified from complex samples by liquid chromatography-mass
spectrometry (LC-MS) analysis. High-pH reversed-phase chromatography is a robust method of peptide fractionation that
separates peptides by hydrophobicity and provides excellent orthogonality to low-pH reversed-phase LC-MS gradients. In
contrast to strong cation exchange (SCX) fractionation, high-pH reversed-phase fractions do not require an additional
desalting step before LC-MS analysis.
The kit includes a high-pH solution (0.1% triethylamine) and 12 spin columns containing pH-resistant, reversed-phase resin.
Each reversed-phase fractionation spin column enables fractionation of 10-100µg of peptide sample using a microcentrifuge.
Native, phosphorylated, Thermo Scientific™ Tandem Mass Tag™ (TMT™)-labeled, and other complex peptide mixture
samples can be fractionated using the kit. Combining the search results generated by the individual fractions improves protein
sequence coverage and increases the number of identified proteins relative to unfractionated samples.
Thermo Fisher Scientific
PO Box 117
(815) 968-0747 or (800) 874-3723
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
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Procedure Summary
Proteolytic digests of proteins extracted from cells or tissues are loaded onto an equilibrated, high-pH, reversed-phase
fractionation spin column. Peptides are bound to the hydrophobic resin under aqueous conditions and desalted by washing the
column with water by low-speed centrifugation. A step gradient of increasing acetonitrile concentrations in a volatile highpH elution solution is then applied to the columns to elute bound peptides into eight different fractions collected by
centrifugation. Each fraction is then dried in a vacuum centrifuge (e.g., Thermo Scientific™ SpeedVac™ Vacuum
Concentrator) and stored until analysis by mass spectrometry. During LC-MS analysis, peptides in each high-pH fraction are
further separated using a low-pH gradient, thus reducing the overall sample complexity and improving the ability to identify
low-abundant peptides.
Figure 1. Spin column conditioning and sample fractionation workflow.
Important Product Information
•
Do not exceed the recommended centrifugation speeds because this may damage the column frit, causing the resin
material to leak, leading to sample loss and/or damage to the LC system.
•
Use low protein-binding microcentrifuge tubes to ensure maximum sample recovery.
•
Store high-pH buffers in polypropylene tubes at room temperature. Do not store high-pH buffers in glass vessels.
•
Avoid sample contamination and direct skin contact with solvents and chemicals. Always wear gloves when handling the
spin columns and samples.
Additional Materials Required
•
Trifluoroacetic acid (Product No. 28904)
•
Acetonitrile (ACN), LC-MS Grade (Product No. 51101)
•
Water, LC-MS Grade (Product No. 51140)
•
Thermo Scientific™ Pierce™ Low Protein Binding Microcentrifuge Tubes, 2.0mL (Product No. 88379 or 88380)
•
Microcentrifuge with adjustable rotor speed up to 7,000 × g
•
Vacuum centrifuge
Thermo Fisher Scientific
PO Box 117
(815) 968-0747 or (800) 874-3723
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
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Material Preparation
•
Trifluoroacetic acid (TFA), 0.1%
Prepare 10mL of equilibration solution by adding 10µL of TFA to 10mL of water.
Volume is sufficient for equilibration of 12 columns.
•
High-pH step-elution solutions
Prepare solutions in 2.0mL tubes according to peptide sample type (Table 1 or
Table 2). Different sets of elution solutions are used for unlabeled, native peptides
or TMT-labeled samples due to different peptide retention behavior. Note that
300µL of each solution is required per sample. Recommended volumes in
Tables 1 and 2 are enough for fractionation of up to three samples.
Table 1. Preparation of elution solutions for unlabeled, native
peptides.
Fraction Acetonitrile Acetonitrile Triethylamine (0.1%)
No.
(%)
(µL)
(µL)
1
5.0
50
950
2
7.5
75
925
3
10.0
100
900
4
12.5
125
875
5
15.0
150
850
6
17.5
175
825
7
20.0
200
800
8
50.0
500
500
Table 2. Preparation of elution solutions for Thermo Scientific
TMT-labeled peptides.
Fraction Acetonitrile Acetonitrile
Triethylamine (0.1%)
No.
(%)
(µL)
(µL)
Wash
5.0
50
950
1
10.0
100
900
2
12.5
125
875
3
15.0
150
850
4
17.5
175
825
5
20.0
200
800
6
22.5
225
775
7
25.0
250
750
8
50.0
500
500
Fractionation of Proteolytic Digests
A. Conditioning of the Spin Columns
Note: Do not exceed recommended centrifugation speeds.
1.
Remove the protective white tip from the bottom of the column and discard. Place the column into a 2.0mL sample tube.
2.
Centrifuge at 5000 × g for 2 minutes to remove the solution and pack the resin material. Discard the liquid.
3.
Remove the top screw cap and load 300µL of ACN into the column. Replace the cap, place the spin column back into a
2.0mL sample tube and centrifuge at 5000 × g for 2 minutes. Discard ACN and repeat wash step.
4.
Wash the spin column twice with 0.1% TFA solution, as described in Step 3. The column is now conditioned and ready
for use.
Thermo Fisher Scientific
PO Box 117
(815) 968-0747 or (800) 874-3723
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
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B. Fractionation of Digest Samples
Note: Each sample requires 300µL of each elution solution. If more than three samples require fractionation, prepare
larger volumes of the elution solutions to accommodate all samples.
1.
Prepare elution solutions according to Table 1 or Table 2 depending on sample type.
2.
Dissolve 10-100µg of digested sample in 300µL of 0.1% TFA solution.
Note: Peptide samples need to be completely dissolved and free of organic solvent (e.g., ACN, DSMO, etc.). If the
sample contains urea, make sure that the final concentration of urea is < 1M.
3.
Place the spin column into a new 2.0mL sample tube. Load 300µL of the sample solution onto the column, replace the
top cap and centrifuge at 3000 × g for 2 minutes. Retain eluate as “flow-through” fraction.
4.
Place the column into a new 2.0mL sample tube. Load 300µL of water onto the column and centrifuge again to collect
the wash. Retain eluate as “wash” fraction.
Note: TMT-labeled samples require an additional column wash with 300µL of 5% ACN, 0.1% TEA (see Table 2) to
remove unreacted TMT reagent.
5.
Place the column into a new 2.0mL sample tube. Load 300µL of the appropriate elution solution (e.g., 5% ACN,
0.1% TEA) and centrifuge at 3000 × g for 2 minutes to collect the fraction.
6.
Repeat Step 5 for the remaining step gradient fractions using the appropriate elution solutions from Table 1 or Table 2 in
new 2.0mL sample tubes.
7.
Evaporate the liquid contents of each sample tube to dryness using vacuum centrifugation (e.g., SpeedVac concentrator).
8.
Re-suspend dry samples in an appropriate volume of 0.1% formic acid (FA) before LC-MS analysis.
9.
Optional: Determine the peptide concentration and yield with a peptide quantitation assay, so equivalent sample
amounts can be analyzed by LC-MS.
Troubleshooting
Problem
Low peptide
yields
Unsuccessful
fractionation
Possible Cause
Low protein yield following
lysis and protein extraction
procedure
Solution
Estimate protein concentration using BCA assay
Lyophilized/dried peptide
samples were not completely
solubilized before sample
loading onto the spin column
Increase vortexing/sonication time to completely dissolve the dried
peptide sample
Incorrect centrifuge speeds
used for fractionation
Ensure proper centrifuge speed is used [in (× g)]. To convert from
revolutions per minute (rpm) to g, use the following formula:
Use an alternative protein extraction procedure
g = (1.118 × 10-5) RS2
where g is the relative centrifugal force, R is the rotor radius in
centimeters, and S is the centrifuge speed in rpm. For example,
centrifugation of a sample at 5,000 rpm in a microcentrifuge having a
rotor radius of 7cm will deliver a centrifugal force of 1,957 × g
Low
peptide/protein
identification
numbers
Low sample load (< 10µg)
Estimate peptide concentration using the Thermo Scientific™ Pierce™
Quantitative Fluorometric Peptide Assay (Product No. 23290) or
Thermo Scientific™ Pierce™ Quantitative Colorimetric Peptide Assay
(Product No. 23275)
Use low protein-binding tubes for handling of the samples and fraction
collection
Incorrect chromatography or
mass spectrometer instrument
settings
Consult instrument user manuals or online resources to determine the
optimal instrument settings for your system
Verify LC-MS system performance with the Thermo Scientific™
Pierce™ HeLa Digest Protein Standard (Product No. 88328)
Thermo Fisher Scientific
PO Box 117
(815) 968-0747 or (800) 874-3723
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
4
thermofisher.com
Related Thermo Scientific Products
90061
TMTsixplex™ Isobaric Label Reagent Set, 1 × 0.8mg
90064
TMTsixplex Isobaric Mass Tagging Kit
90110
TMT10plex™ Isobaric Label Reagent Set, 1 × 0.8mg
90113
TMT10plex Isobaric Mass Tag Labeling Kit, 30 rxns
90101
iodoTMTsixplex™ Label Reagent Set, 1 × 0.2mg
90103
iodoTMTsixplex Isobaric Mass Tag Labeling Kit
90401
aminoxyTMTsixplex™ Label Reagent Set, 1 × 0.2mg
84840
Pierce™ Mass Spec Sample Prep Kit for Cultured Cells
88328
Pierce HeLa Digest Protein Standard
23227
BCA Protein Assay Kit
23275
Pierce Quantitative Colorimetric Peptide Assay
23290
Pierce Quantitative Fluorometric Peptide Assay
90057
Pierce™ Trypsin Protease, MS Grade
28904
Trifluoroacetic Acid, Sequanal Grade
51140
Water, LC-MS Grade
51101
Acetonitrile (ACN), LC-MS Grade
General References
Feng Yang, Yufeng Shen, David G. Camp II, Richard D. Smith (2012). High pH reversed-phase chromatography with fraction concatenations as an
alternative to strong-cation exchange chromatography for two-dimensional proteomic analysis. Expert Rev Proteomics 9(2) 129-34.
Mohammad Pirmoradian, Harshavardhan Budamgunta, Konstantin Chingin, Bo Zhang, Juan Astorga-Wells, Roman A. Zubarev (2013). Rapid and deep
human proteome analysis by single-dimension shotgun proteomics. Mol Cell Proteomics 12(11) 3330-8.
Washburn, M.P.; Wolter, D.; Yates, J.R. 3rd (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat
Biotechnol 19(3) 242-7.
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Thermo Fisher Scientific
PO Box 117
(815) 968-0747 or (800) 874-3723
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
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