Automation of Phosphoenrichment using Magnetic Fe-NTA Beads and KingFisher™ Apex Magnetic Particle Processor
Maureen Mccoy*; Amarjeet Flora M.S.**; Leigh Foster B.S.**; Penny Jensen Ph.D.**; Bhavin Patel MD, M.S.**; Sergei Snovida Ph.D.**; Ryan Bomgarden Ph.D.**
*University of Illinois Urbana Champaign **Thermo Fisher Scientific, Rockford, IL
ABSTRACT
Purpose: Phosphorylation is a critical post translational modification that modulates the function of
numerous proteins and recent advances in the mass spectrometry (MS) instrumentation have
enabled studying phosphorylation at proteomics scale in complex biological samples. Due to the low
stoichiometry of phosphorylation in biological samples, IMAC has been widely used for enriching
phosphorylated peptides. Here, we introduce an agarose-based Fe-NTA magnetic bead for manual
and automated phosphopeptide enrichment workflows using Thermo Scientific™ Kingfisher™ Apex
Magnetic Particle Processor for high throughput applications.
Methods: Nocodazole treated HeLa S3 cells were processed using Thermo Scientific™ EasyPep™
Maxi MS Sample preparation kit. Magnetic Fe-NTA beads were incubated with protein digests and
magnetically separated from the supernatant manually or through automation using Kingfisher Apex
Magnetic Particle Processor for the phosphopeptide enrichment. Peptides were quantified and
normalized using the Thermo Scientific™ Pierce™ Quantitative Colorimetric Peptide Assay prior to
LC-MS analysis using a Thermo Scientific™ Orbitrap™ Q Exactive™ Plus mass spectrometer.
Thermo Scientific™ Proteome Discoverer™ 2.4 software was used to localize the phosphorylation
sites.
Results: Our optimized EasyPep chemistry combined with the large-scale format and subsequent
phosphopeptide enrichment provides a complete workflow solution in less than 7 hours. We have
identified ~8000-9000 phospshopeptides with ~95% phosphospecificity and CVs <5%. We have
compared it to the existing resin workflows and observed identical performance in terms of
phosphopeptide specificity and identification rates. We have also assessed the workflow on a
Kingfisher Apex Magnetic Particle Processor which ensures reproducibility and eliminates the hands-
on-challenges while handling a large number of samples.
INTRODUCTION
Phosphorylation is a post translational modification (PTM) that acts as a molecular switch in cell
signaling pathways. Because phosphorylation misregulation is key factor in many human diseases,
high throughput studies that identify changes in phosphorylation are important for understanding
disease mechanisms. In these studies, reducing variability between samples is key. For studying the
phosphoproteome, additional enrichment is required before mass spec analysis due to the lability and
low stoichiometry of phosphorylation. This enrichment can be done using Immobilized Metal Affinity
Chromatography (IMAC) with Fe-NTA which has selectivity for the phosphate group at the
appropriate acidic pH. The purpose of this study was to use Fe-NTA magnetic agarose beads to
optimize a Thermo Scientific™ KingFisher ™ Apex protocol for high-throughput phosphoenrichment.
MATERIALS AND METHODS
Sample Preparation
HeLa S3 cells were cultured in sMEM media supplemented with 10%FBS, 1X Glutamax, and 1%
Pen/Strep. Cells were treated with Nocodazole at 0.1µg/ml for 18 hours. HeLa S3 harvested cells and
CSF samples were processed into protein digests with Thermo Scientific™ EasyPep™ Maxi MS
Sample preparation kit and Halt phosphatase inhibitor. Protein concentrations were determined using
Pierce™ Rapid Gold BCA Assay kit. CSF samples were additionally labelled with Thermo Scientific™
TMTpro™ 16plex reagents .
Phosphoenrichment
KingFisher Apex Magnetic Particle Processor, KingFisher 96 Deep-Well plates, and Fe-NTA magnetic
agarose beads were used for phosphopeptide enrichment. The digests were incubated with Fe-NTA
magnetic beads, organic and aqueous wash steps were performed, and peptides were eluted in a
basic buffer. Bead to digest ratios, elution times, wash volume, and plate rinses were tested to
optimize the protocol. Peptide desalting using a mixed mode resin in a tip was done to reduce
possible contaminants. For a positive control, manual phosphoenrichment was done using Fe-NTA
magnetic beads on a magnetic stand.
LC-MS Analysis
Before LC-MS analysis, peptide concentration was determined using Thermo Scientific™ PierceTM
Quantitative Colorimetric Peptide Assay kit. Samples were separated using a Thermo Scientific™ Dionex™
Ultimate™ 3000 Nano LC system using a 50 cm C18 Thermo Scientific™ EASY-Spray™ column with an
acetonitrile gradient from 3% to 28% over 85 min, 28% to 45% over 30 min, at a flow rate of 300nL/min on a
Thermo Scientifc™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer. CSF Samples were
analyzed on an Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer. To identify plastic
contaminants, Fe-NTA enrichment buffers were incubated on KingFisher 96 Deep-Well plates and directly
injected into a Thermo Scientific™ QExactive™ HF Hybrid Quadrupole-Orbitrap™ mass spectrometer
CONCLUSIONS
KingFisher automation of phosphoenrichment with Fe-NTA magnetic beads has low variability
between samples with up to 10,000 phosphopeptide ID’s and 95% phosphopeptide specificity and is
comparable or improved to manual magnetic phosphoenrichment.
KingFisher Apex automation was optimized with cleanup or an elution plate rinse, a 1:50 bead to
sample digest ratio, a one minute elution time, and a 100µl wash volume.
Application of this protocol to CSF samples resulted in 202 more phosphopeptide ID’s and
identification of phosphorylated proteins of interest in disease.
REFERENCES
Dunn, Jamie D., et al. “Techniques for Phosphopeptide Enrichment Prior to Analysis by Mass
Spectrometry.” Mass Spectrometry Reviews, vol. 29, no. 1, Feb. 2010, pp. 29–54. PubMed,
https://doi.org/10.1002/mas.20219.
Timpl, Rupert, et al. “Fibulins: A Versatile Family of Extracellular Matrix Proteins.” Nature Reviews
Molecular Cell Biology, vol. 4, no. 6, June 2003, pp. 479–89. www.nature.com,
https://doi.org/10.1038/nrm1130.
Automation of Phosphoenrichment using Magnetic Fe-NTA Beads and
KingFisher™ Apex Magnetic Particle Processor
HeLa S3 Cells
EasyPepTM Sample Prep:
Lysis, Reduction,
Alkylation & Digestion
EasyPepTM
Cleanup
KingFisherTM Apex Phosphopeptide
Enrichment
Analyze with Thermo
ScientificTM Proteome
Discoverer 2.4 Software
Nocodazole-
Treated
Cell Pellet
Peptides + Contaminants
Clean Peptides
Phosphopeptides
Figure 4: Evaluation of Leachables from KingFisherTM Plastic Consumables
KingFisher plastics are designed for compatibility with DNA and RNA workflows and interaction with enrichment solvents
leads to leaching of contaminants. Direct injection of a blank onto the Q Exactive™ HF mass spectrometer shows an
acceptable number of contaminant peaks. Injection of acetonitrile and ammonium hydroxide incubated on KingFisher 96
Deep-Well plate at 4oC overnight shows an increased number of peaks consistent with plasticizers and detergents listed
in MS contaminant databases. After several rounds of sample and blank injections, the contaminant peaks remaining in
the final blank indicate the persistence of these contaminants and the need for extended flushing.
pos_blank_01 #1 RT: 0.00 AV: 1 NL: 1.16E8
T: FTMS + p ESI Full ms [100.0000-2000.0000]
100
150
200
250
300
350
400
450
500
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ati
ve A
bundanc
e
338.34
227.18
453.34
475.32
498.40
149.02
244.19
310.31
431.38
114.09
371.10
415.21
171.99
223.06
297.08
352.32
272.23
511.47
491.30
533.19
332.33
Blank
pos_Plate1A_H1_repeat #1 RT: 0.00 AV: 1 NL: 9.85E7
T: FTMS + p ESI Full ms [100.0000-2000.0000]
150
200
250
300
350
400
450
500
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ati
ve A
bundanc
e
331.28
236.11
348.31
359.31
313.27
376.34
159.06
239.24
228.20
404.37
432.24
267.27
391.28
114.09
303.25
135.10
171.99
476.20
539.21
460.27
205.09
515.21
N-butyl
benzenesulfo
namide
(236.11)
Triton
(331.22)
Dicyclohexyl
phthalate
(348.22)
Acetonitrile Incubation on
KingFisher DeepWell Plate
Figure 3: Current Solutions for Plastic Leaching
A) Evaluating post-enrichment cleanup: 500µg of EasyPep processed peptides (1µg/µl) were incubated with beads in a
1:50 ratio with a wash volume of 100µl and a one minute elution time. After enrichment, cleaned samples were desalted
on a mixed mode resin. 500ng of enriched samples and 500ng of the Fe-NTA magnetic manual comparison sample were
analyzed by LCMS as described in the methods. Including cleanup improved ID’s by more than 4-fold and percent
phosphospecificity was high (~97%). The disadvantages of cleanup include decreased yield and efficiency.
B) Evaluating
an elution plate rinse: 500µg of EasyPep processed peptides (1µg/µl) were incubated with beads in a 1:50 ratio with a
wash volume of 100µl and a one minute elution time. Elution plate wells were either rinsed twice with acetonitrile or left
empty before adding elution buffer and performing enrichment. 750ng of enriched samples and 500ng of the Fe-NTA
magnetic manual comparison sample were analyzed by LCMS as described in the methods. Including a rinse improved
phosphopeptide ID’s by nearly two-fold and phosphopeptide specificity remained high (~96%).
C) Images of the above
rinse/no rinse samples that have undergone vacuum centrifugation for the same time period. Comparing with the sample
on the right, decreased color and faster drying time indicates a decrease in plastic leachables with rinsing.
92.8
96.1
98.3
85
87
89
91
93
95
97
99
101
Manual
Rinse
No Rinse
Per
cent
S
pec
ifi
ci
ty
Average % Phosphopeptide Specificity
2123.5
2565
1345.5
0
1000
2000
3000
4000
Manual
Rinse
No Rinse
Phos
phopept
ide
ID
’s
Average Phosphopeptide ID's
With Rinse
Without Rinse
A)
C)
B)
3558.5
4291.7
793.7
0
1000
2000
3000
4000
5000
Manual
Cleanup
No Cleanup
Phos
phopept
ide
ID
’s
Average Phosphopeptide ID's
90.8
97.1
95.4
85
90
95
100
Manual
Cleanup
No Cleanup
Per
cent
S
pec
ifi
ci
ty
Average % Phosphopeptide Specificity
Figure 5: Optimization of Bead to Sample Digest Ratios
Using the Thermo Scientific™ KingFisher™ Apex, 500µg of EasyPep processed peptides (1µg/µl) were incubated with
beads in a 1:10, 1:20 or 1:50 ratio. 500ng of enriched samples and 500ng of the Fe-NTA magnetic manual sample were
analyzed by LC-MS as described in the methods. The results show that a 1:50 bead to sample ratio maximizes
phosphopeptide specificity with only a minor loss in phosphopeptide ID’s.
11875
9935
11056
10506
0
4000
8000
12000
16000
1:40
1:10
1:20
1:50
Manual
KingFisher
Phos
phopept
ide
ID
’s
Average Phosphopeptide ID's
91.0
84.2
91.4
95.5
0
20
40
60
80
100
120
1µg Mag
Beads
1:10Ratio
1:20Ratio
1:50Ratio
Manual
KingFisher
Phos
phopept
ide
Spec
ifi
ci
ty
Average %Phosphopeptide Specificty
Figure 6: Optimization of Wash Volume
Using the Thermo Scientific™ KingFisher™ Apex, 500µg of EasyPep processed peptides (1µg/µl) were incubated with
beads in a 1:50 bead to sample ratio. Organic wash volumes of 500µl or 100µl were compared. 500ng of enriched
samples and 500ng of the Fe-NTA magnetic manual comparison sample were analyzed by LC-MS as described in the
methods. 100µl wash volume slightly improved phosphopeptide ID’s and percent phosphospecificity with greater
reproducibility.
3,558.5
3,379.7
4,291.7
0
1000
2000
3000
4000
5000
Mag Beads
500µl Wash
100µl Wash
Phos
phopept
ide
ID
’s
Average Phosphopeptide ID's
90.8
93.0
97.1
0
20
40
60
80
100
120
Mag Beads
500µl Wash
100µl Wash
Per
cent
S
pec
ifi
ci
ty
Average %Phosphopeptide Specificty
Figure 7: Cerebrospinal Fluid Sample Prep and LCMS Analysis Workflow
Inject into Thermo
Scientific™ Orbitrap
Eclipse™ Tribrid™ mass
spectrometer
Normal 1
Normal 2
Alzheimer’s Disease
Mild Cognitive
Impairment
Parkinson’s disease
EasyPepTM Sample Prep:
Lysis
Phosphatase Inhibition
Reduction/Alkylation
Digestion
Label with
TMTproTM 16 plex
Pool all samples
Optimized KingFisherTM
Apex Phosphopeptide
Enrichment
EasyPepTM Cleanup
Phosphopeptides
Analyze with Thermo Scientific™ Proteome
Discoverer™ 2.4 Software
Enriched
Sample
Figure 8: Application of Phosphoenrichment to Cerebrospinal Fluid Samples
CSF samples from two normal patients, one Alzheimer's Disease, one Mild Cognitive Impairment, and one Parkinson’s
patient were processed according to the unenriched and enriched workflows in Figure 6. A) Phosphopeptide ID’s
increased in the enriched sample from 25 to 227 with a specificity of 54%. B) PCA plot showing reproducibility with each
samples appropriately showing as a distinct population. C) Abundance of Fibulin-1 Isoform A in phosphoenriched CSF
samples. Fibulin-1 was found to be phosphorylated in the enriched samples but not in unenriched samples. Although not
differentially phosphorylated, Fibulin-1 has lower expression in diseased samples which may correspond with Fibulin’s
ability to bind the N-terminus of APP – a protein implicated in AD2.
25
227
0
50
100
150
200
250
Unenriched
Enriched
Phos
phopept
ide
ID
’s
Phosphopeptides ID's
0.6
54.4
0
10
20
30
40
50
60
Unenriched
Enriched
Per
cent
S
pec
ifi
ci
ty
% Phosphospecificty
Alzheimer’s
MCI
Normal 2
Normal 1
Parkinson’s
Pool
Alzheimer’s
MCI
Normal 1
Normal 2
Parkinson’s Pool
A)
C)
B)
RESULTS
Figure 1: Workflow for Phosphopeptide Enrichment
TRADEMARKS/LICENSING
For Research Use Only. Not for use in diagnostic procedures or protocols.
© 2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher
Scientific and its subsidiaries. This information is not intended to encourage use of these products in any
manner that might infringe the intellectual property rights of others.
PO66115 EN0921S
Figure 2: Evaluation of Magnetic Beads for Phosphopeptide Enrichment
A). Magnetic beads were evaluated for total binding capacity versus the Thermo Scientific™ High-Select™ Fe-NTA
Phosphopeptide Enrichment Kit and three competitor products used for phosphopeptide enrichment. 15ug of Beta Casein
monophosphopeptide was allowed to bind to a fixed, equivalent amount of resin or bead for all samples. Protocols were
followed according to manufacturer’s instructions. Thermo Scientific™ Pierce™ Phosphoprotein Phosphate Estimation
Assay Kit was used to quantitate the amount of protein remaining in the flow through after binding. Total amount of Beta
Casein Monophosphopeptide bound was calculated by subtracting this value from the initial load.
B) HeLa S3 cells treated
with Nocodazole, were processed using EasyPep workflow, spiked with 131 Heavy AQUA phosphopeptides, and
subjected to phosphoenrichment with either Thermo Scientific™ High-Select™ Fe-NTA Phosphopeptide Enrichment Kit
(with or without SMOAC method), Magnetic Fe-NTA beads, or Competitor Beads A, which performed well in initial binding
capacity assessments. Samples were analyzed by Mass spec for number of phosphopeptides versus total peptide
identifications as well as targeted analysis to evaluate recovery of heavy and light peptides of interest in a complex matrix.
A)
B)
12.6
11.7
5.0
2.6
0
4
8
12
16
Thermo
Scientific
Competitor A
Competitor B
Competitor C
Be
ta
-C
as
ei
n
M
onophos
phopept
ide
B
ound
(ug)
Magnetic Bead Benchmark for Phosphoenrichment
Binding Capacity
7121.5
6387
7044
6974.5
0
1000
2000
3000
4000
5000
6000
7000
8000
Thermo
SMOAC
Method
Thermo
Agarose
Thermo
Magnetic
Competitor
A
#
Phos
phopept
ides
Ident
ifi
ed
Phosphoenrichment Benchmark: Number of
Phosphopeptides
97%
89%
94%
88%
0%
20%
40%
60%
80%
100%
Thermo
SMOAC
Method
Thermo
Agarose
Thermo
Magnetic
Competitor
A
Phos
phopept
id
e S
pec
ifi
ci
ty
Phosphoenrichment Benchmark: Percent
Phosphospecificity
94.2%
91.4%
95.7%
92.1%
31.7%
25.2%
31.7%
25.2%
0%
20%
40%
60%
80%
100%
Thermo SMOAC Method
Thermo Agarose
Thermo Magnetic
Competitor A
Pept
ide
Peak
A
rea
PRM Acquisition of Phospho-enriched Heavy Peptides in Nocodazole-Treated HeLa
Heavy
Peptides
Light
Peptides
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