Examining ubiquitinated peptide enrichment efficiency through an epitope labeled protein

Article abstract of DOI:10.1016/j.ab.2016.08.017

Ubiquitination is a dynamic process that is responsible for regulation of cellular responses to stimuli in a number of biological systems. Previous efforts to study this post-translational modification have focused on protein enrichment; however, recent r

Full text of DOI:10.1016/j.ab.2016.08.017

Accepted Manuscript
Examining ubiquitinated peptide enrichment efficiency through an epitope labeled
protein
J. Parker, Y. Oh, Y. Moazami, J.G. Pierce, P.L. Loziuk, R.A. Dean, D.C. Muddiman
PII:
S0003-2697(16)30255-X
10.1016/j.ab.2016.08.017
YABIO 12482
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 14 February 2016
Revised Date: 2 July 2016
Accepted Date: 19 August 2016
Please cite this article as: J. Parker, Y. Oh, Y. Moazami, J.G. Pierce, P.L. Loziuk, R.A. Dean, D.C.
Muddiman, Examining ubiquitinated peptide enrichment efficiency through an epitope labeled protein,
Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.08.017.
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Examining Ubiquitinated Peptide Enrichment Efficiency through an Epitope Labeled Protein
Parker, J.1,2, Oh, Y.3,4, Moazami, Y.1, Pierce, J. G.1, Loziuk, P.L.1,2, Dean, R. A.3,4, Muddiman,
D.C.1,2*
1. Department of Chemistry, North Carolina State University, Raleigh, North Carolina
2. W. M. Keck FTMS Laboratory for Human Health Research, North Carolina State
University, Raleigh, North Carolina
3. Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina
4. Center for Integrated Fungal Research, North Carolina State University, Raleigh, North
Carolina
Subject Category: Mass Spectrometry
*Corresponding author:
Email address: dcmuddim@ncsu.edu
2620 Yarborough Dr, Raleigh, NC 27695

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Abstract
Ubiquitination is a dynamic process that is responsible for regulation of cellular responses to
stimuli in a number of biological systems. Previous efforts to study this post-translational
modification have focused on protein enrichment; however, recent research utilizes the
presence of the di-glycine (Gly-Gly) remnants following trypsin digestion to immuno-enrich
ubiquitinated peptides. Monoclonal antibodies developed to the cleaved ubiquitin modification
epitope, (tert-butoxycarbonyl) glycylglycine (Boc-Gly-Gly-NHS)1, are used to identify the Gly-Gly
signature. Here, we have successfully generated the Boc-Gly-Gly-NHS modification and
showed that when conjugated to a lysine containing protein, such as lysozyme, it can be applied
as a standard protein to examine ubiquitinated peptide enrichment within a complex
background.
Keywords:
•
•
•
Ubiquitination
Gly-Gly Enrichment
Enrichment Efficiency
1. Introduction
The process of ubiquitination occurs through the E3 ligase conjugation of ubiquitin to ε-amines
of lysine, and is known to be a post-translational modification in living organisms that is used to
regulate protein activity in response to a stimulus [1, 2]. Progress in the area of ubiquitomics has
evolved to include antibody recognition of the K-ε-Gly-Gly group that remains after cleavage
with the endoproteinase trypsin [3]. Using the epitope Boc-Gly-Gly-NHS conjugated to histone,
researchers were able to generate the Gly-Gly antibody and subsequently use it for Gly-Gly
peptide enrichment [3]. Kits that utilize anti-Gly-Gly are now commercially available, and have
been shown to be successful in capturing up to thousands of ubiquitinated peptides, providing
new information on dimensions of the proteome that were previously untouched by researchers
[4, 5]. However, when trying to optimize experimental conditions, resulting changes in the
enrichment process cannot always be attributed to be the result of the applied experimental
variable, as variability in handling during enrichment is possible. Therefore, changes in the
enrichment process due to human error must be ruled out. The use of synthetic peptides can be
used to examine enrichment efficiency as well as disassociation bias during LC-MS/MS analysis
[6], but an epitope labeled protein, spiked in before digestion, enables researchers to track each
step of the sample handling process from digestion to LC-MS/MS.
1 Abbreviations used: Boc-Gly-Gly-NHS, (tert-butoxycarbonyl) glycylglycine; Gly-Gly, di-glycine;
T3P®, propylphosphonic anhydride solution; DMSO, dimethyl sulfoxide; TFA, trifluoroacetic acid;
LC, liquid chromatography; MS/MS, tandem mass spectrometry; NMR, nuclear magnetic
resonance; FASP, filter aided sample preparation; DTT, dithiothreitol; NEM, N-ethylmaleimide;
DMP, dimethylpimelimidate; MOPS, 3-(N-morpholino)propanesulfonic acid; IAP, immunoaffinity
purification; PBS, phosphate buffered saline; FA, formic acid; ACN, acetonitrile; AGC, automatic
gain control; FDR, false discovery rate

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To the best of our knowledge, there is no evidence of the application of an epitope labeled
protein being used to measure ubiquitinated peptide enrichment efficiency. The need for such a
modified protein encouraged us to generate the epitope Boc-Gly-Gly-NHS [3, 7-9] and
conjugate this to lysozyme in order to generate an epitope labeled protein standard with the
same chemical modification as the peptides of interest. Because the modified protein enters the
experimental design at the beginning of the sample workflow, one can examine if experimental
procedures hinder the enrichment process by calculating the enrichment efficiency of the protein
standard. Here we show that even when spiked in at low concentrations into a complex
background, we were able to track a modified peptide through the Gly-Gly enrichment
procedure and successfully calculate enrichment efficiency.
2. Materials and Methods
2.1 2,5-diOxopyrrolidin-1-yl-(tert-butoxycarbonyl)glycylglycinate synthesis
To a solution of Gly-Gly (1.00 g, 7.57 mmol) in
dioxane:water (30:5 mL) at room temperature
was added triethylamine (1.15 g, 11.4 mmol)
and di-tert-butyl-dicarbonate (1.84 g, 8.33
mmol) consecutively. The mixture was stirred at
room temperature overnight, then diluted with
Fig. 1 . Chemical structure of (tert-
butoxycarbonyl) glycineglycine
water, acidified to approximately pH = 2 via the
addition of solid KHSO4, extracted with EtOAc,
dried (Na2SO4), and concentrated in vacuo to
afford (tert-butoxycarbonyl)glycylglycine [8] as a white solid. To a solution of crude (tert-
butoxycarbonyl)glycylglycine (0.106 g, 0.451 mmol) in anhydrous dicholoromethane (5.00 mL)
was added triethylamine (45.6 mg, 0.451 mmol) and propylphosphonic anhydride solution
(T3P®) [9] (0.344 g, 0.541 mmol) at room temperature. The mixture was stirred for 20 min,
followed by the addition of N-hydroxy succinimide (51.9 mg, 0.451 mmol). The reaction was
stirred for 48 h and upon completion, the organic layer was washed with brine (3×), dried
(Na2SO4), and concentrated in vacuo to afford the product as a white solid. The crude solid was
triturated from diethyl ether to afford 65.4 mg (44%) of the product as a white solid: 1H NMR
(400 MHz, CDCl3) δ 6.76 (brs, 1 H), 5.15 (brs, 1 H), 4.43 (d, 2 H, J = 5.7 Hz), 3.87 (d, 2 H, J =
5.3 Hz), 2.85 (s, 4 H), 1.45 (s, 9 H); ESIMS m/z 330 [M+H]+. Direct infusion ESI-MS on a high
resolution accurate mass measurements (Exactive Plus, Thermo Fisher Scientific) in the
negative ion mode yielded an elemental composition of the expected product within 5 ppm.
Reactions were monitored by LC-MS (Shimadzu LC-MS 2020 with Kinetex 2.6 mm C18 50 ×
2.10 mm). 1H and 13C NMR spectra were obtained on a Varian Mercury-VX 300, a Varian
Mercury-VX 400, or a Varian Mercury-Plus 300 instrument in CDCl3 unless otherwise noted.
Chemical shifts were reported in parts per million with the residual solvent peak used as an
1
internal standard (CDCl3 H δ = 7.26 and 13C δ = 77.23). 1H NMR spectra were run at 300 or
400 MHz and are tabulated as follows: chemical shift, multiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, dd = doublet of doublet, qd = quartet of doublets, ddt = doublet
of doublet of triplet), number of protons, and coupling constant(s).
All reactions were performed under an Ar atmosphere and all glassware was dried in an oven at
135 °C overnight prior to use, unless otherwise not ed. Dichloromethane was purified using an

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alumina filtration system. Diglycine, triethylamine, di-tert-butyl-dicarbonate, N-hydroxy
succinimide, and T3P® were purchased from Sigma Aldrich and Fisher Scientific and used as
received unless otherwise noted.
2.2 Lysozyme modification
Gly-Gly-modified lysozyme was generated by following the protocol described in Xu et al [3]. In
brief, 1 mg of lysozyme (Sigma, CAS# 12650-88-3) was dissolved in 100 mM NaHCO3 buffer
(2.5 mL) at pH 10. 125 µL of 50 mM, Boc-Gly-Gly-NHS in dimethyl sulfoxide (DMSO) was
added to protein solution and the reaction was carried at 25 °C for 1 h by constant shaking (200
rpm) on a plate rotator. This reaction was repeated three times. For de-protection of Boc group
from the modified protein, 1.5 mL trifluoroacetic acid (TFA) was added, and the solution was
shaken for 2 h at 25 °C. After neutralized with 10 M NaOH dropwise on ice, the reaction was
dialyzed (8-10 MWCO, Spectrum Laboratories) four times (1 h 3 times at 25 °C and overnight at
4 °C) against 20 mM acetic acid (3 L).
2.3 Western blot analysis
To confirm the Gly-Gly modification of lysozyme, we followed the protocols previously published
by Xu et al [3]. Briefly, 5 µg of each unmodified lysozyme, Gly-Gly modified, and Boc-Gly-Gly
modified lysozyme samples were separated on a 4–20% gradient gel (Invitrogen) and
transferred to a nitrocellulose membrane (Bio-Rad). Blots were blocked in 5% bovine serum
albumin and antibody incubations were carried out in 5% skim milk followed by washes. Signals
were detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce), following the
manufacturer’s instructions. The following antibodies were used: Gly-Gly lysine hybridoma clone
GX41 (1:2000, Lucerna) and Anti-mouse IgG conjugated to horseradish peroxidase (1:3000,
Cell Signaling Technology).
2.4 Sample preparation and protein extraction
The complex background proteome was generated from mycelia collected from a M. oryzae
strain 70-15 culture. Spore harvest and inoculation was performed as in Oh et al [10]. The
mycelia were treated with nitrogen limiting minimal media (10 g sucrose, 1 mL A. nidulans trace
elements, 1 g thiamine, and 5 ꢀg biotin in 1 L). Proteins were extracted using lysis buffer ( 8M
urea, 50mM Tris-HCl, pH 7.5, 150mM NaCl, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride
(PMSF), 50uM PR-619, Protease Inhibitor cocktail (Roche)) [11]. Protein concentration was
measured by Bradford assay.
2.5 Sample digestion
2.5.1 Lysozyme
Lypholized epitope modified lysozyme was re-suspended in 0.001% Zwittergent 3-16
(Calbiochem, La Jolla, CA), and a 1:1 combination of modified to unmodified lysozyme was
generated from 0.001% Zwittergent reconstituted epitope modified lysozyme and unmodified
lysozyme. The protein standard mixture was spiked in at 0.1%, 1%, and 5% (mg lysozymel/mg
Magnaporthe oryzae proteins) using total proteins extracted from the fungus Magnaporthe
oryzae (M.oryzae) as the complex protein background. All samples were digested using filter
aided sample preparation (FASP) [12]. Each sample was diluted 2-fold with 100 mM
dithiothreitol (DTT) in 8 M urea, 50 mM Tris, pH 7.0 and incubated at 56 °C for 30 min. Samples

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were alkylated with a final concentration of 50 mM chloroacetamide in 8 M urea, 50 mM Tris, pH
7.0 at 37 °C for 60 min. The alkylated sample was t hen transferred to a 10 kDa centrifugal filter
(Millipore, #UFC501096) and concentrated by centrifuging at 14,000 × g at 20 °C, 15 min. The
filter containing the sample was then washed 3 times with 2 M urea, 10 mM CaCl2 in 50mM
Trish pH 7.0. Each sample was trypsin digested at a 1:50 enzyme:protein ratio. The digestion
reaction was incubated overnight at 37 °C. The reac tion was quenched with 400 uL of 1%
formic acid in 0.001% Zwittergent, and subsequently centrifuged at 14,000 × g at 20 °C, 15 min
to elute the peptides.
2.5.2 Magnaporthe oryzae
Samples prepared for enrichment were digested and enriched as in Porras-Yakushi et al [6],
with minor modifications. A total of 10 mg of protein extracted from treated M. oryzae was used
for Gly-Gly enrichment. The sample was cleared by centrifugation at 16000 × g for 15 min.
Before the digestion process, 1:1 ratio of modified: unmodified protein standard was spiked in at
0.1%. The sample was reduced for 45 min at room temperature with a final concentration of 4.5
mM DTT. Following reduction, the sample was alkylated with final concentration of 10 mM N-
ethylmaleimide (NEM). This was performed for 30 min in the dark at room temperature. LysC
digestion was carried out at a ratio of 1:200 at room temperature. Following the first digestion,
the sample was diluted to 2 M urea with 100 mM Tris (pH 8.0). CaCl2 was added to the sample
for a final concentration of 1 mM, and subsequent trypsin digestion at a ratio of 1:100 was
performed at 37 °C overnight.
The digestion reaction was quenched with 0.1% TFA in H2O and concentrated in vacuo.
Following centrifugation at 16000 × g for 15 min to clear the sample, desalting was performed
using the 500 mg SepPak column (Waters). The column was washed with 21 mL of 100%
acetonitrile (ACN) and equilibrated with 21 mL of equilibration buffer (0.1% TFA, in H2O). The
dried peptides were re-suspended in equilibration buffer and loaded onto the SepPack column.
A 21 mL wash with the same buffer was performed and followed with a wash with 9 mL wash
with 0.1% TFA, 5% ACN in H2O. Peptides were eluted with 6 mL of 0.1% TFA, 40% ACN in
H2O.
2.6 Resin cross-linking and Gly-Gly enrichment
For the chemical cross-linking of antibody to bead, we followed protocols previously published
by Udeshi et al and Porras-Yakaushi et al [6, 11]. Briefly, the antibody-bound beads were
washed three times with 1 mL of antibody cross-linking wash buffer (100 mM sodium borate, pH
9.0). The antibody beads were re-suspended in 1 mL of antibody cross-linking buffer (20 mM
dimethylpimelimidate (DMP) in 100 mM sodium borate, pH 9.0) and incubated in gentle end-
over-end rotator for 30 min at room temperature. The cross-linking reaction was stopped by
washing the antibody beads twice with 1 mL of antibody blocking buffer (200 mM ethanolamine,
pH 8.0). The antibody beads were re-suspended with 1 mL antibody blocking buffer and
incubated for 2 h at 4 °C with gentle rotation. The cross-linked antibody was washed three times
with 1.5 ml of IAP buffer (50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.2), 10
mM Na3PO4 and 50 mM NaCl).
De-salted peptides were dried down with a speed vacuum and subsequently re-suspended in 1
mL 1× immunoaffinity purification (IAP) buffer (50 mM MOPS (pH 7.2), 10 mM sodium
phosphate, and 50 mM NaCl). At this point, 100 µg of sample was removed and set aside for a
non-enriched sample. After centrifuging at 20000 × g for 5 min, the pH was adjusted to 7.0 by

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adding 1 M Tris pH 7.0. Re-suspended peptides were added to the washed cross-linked
antibody resin and incubated with end-over-end rotation for 1 h at 4 °C [6, 11]. After incubation,
the flow through was removed, and the resin was washed three times with 500 µL 1 × IAP
buffer, centrifuging at 2000 × g for 1 min each time. One wash with 1 × phosphate-buffered
saline (PBS) followed by one wash with H2O was performed, centrifuging at 2000 × g for 1 min
after each wash. Peptides were eluted with 150 µL 0.15% TFA.
Samples were cleaned up using C18 ZipTips (Millipore). The dried enriched peptides were re-
suspended in 15 µL of 0.1% formic acid (FA). The ZipTip was washed with 9 µL of 100% ACN,
and equilibrated with 90 µL of 0.1% FA, H2O. The sample was loaded onto the ZipTip by
drawing up the sample and expelling it slowly repeatedly for 2 min. The loaded sample was
washed with 100 µL 0.1% FA and subsequently eluted with 25 µL 80% ACN, 20% 0.1% FA.
The sample was dried before preparing for LC-MS/MS analysis.
2.7 LC-MS/MS
2.7.1 Lysozyme
All samples were separated using the Easy nanoUPLC (Thermo) and analyzed on a Q Exactive
High Field (Thermo). Columns were Self-Pack PicoFrit Columns 360 µm O.D. 75 µm I.D. 15 um
tip I.D. with no coating (New Objective Inc. PF360-75-15-N-5). Columns were packed with 2.6
µm C18 100 Å Kinetix packing material (Phenomenex). Mobile phase A consisted of 98% H2O,
2% ACN, and 0.2% FA; Mobile phase B consisted of 2% H2O, 98% ACN, and 0.2% FA.
Both the Gly-Gly epitope modified lysozyme and the protein standard 1:1 mixture were loaded
onto a 25 cm packed column and eluted at a flow rate of 300 nL/min with a 30 min linear
gradient (5-30%). Full MS was acquired with a resolving power of 60,000, an automatic gain
control (AGC) target of 1e6 and a scan range 300-2000 m/z. During data dependent MS2, a
15,000 resolution was applied with an AGC target of 2e4. Maximum injection time was set to 30
ms for both MS1 and MS2. A total of 12 sequencing events were allowed per scan. A 2 m/z
isolation window was utilized with normalized collision energy of 27and a dynamic exclusion of
30 sec.
2.7.2 Enriched, flow through, and non-enriched samples
The 0.1% spiked in control 1:1 protein in the M. oryzae background as well as the enrichment
samples (enriched peptides, flow through peptides, and non-enriched peptides) were loaded
onto a 20 cm packed column and eluted at a flow rate of 300 nL/min with a 120 min linear
gradient. The control sample was analyzed using a 5-30% gradient of mobile phase B, and the
enrichment, flow through, and non-enriched samples were analyzed using a 5-32% gradient.
Full MS was acquired with a resolving power of 120,000, an AGC target of 3e6, a scan range
300-1600 m/z, and a maximum injection time of 50 ms. During data dependent MS2, a 15,000
resolution was applied with an AGC target of 1e5 and a maximum injection time of 30 ms. A
total of 20 sequencing events were allowed per scan. A 2 m/z isolation window was utilized with
normalized collision energy of 27, and a dynamic exclusion of 20 sec.
2.8 Database searching
RAW files were submitted to Proteome Discoverer 1.4 and using SEQUEST HT, searched
against a targeted M. oryzae database of 13,043 sequences [13]. Trypsin (Full) was selected for
the digestion enzyme, and 4 maximum missed cleavage sites were allowed. A precursor mass

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tolerance of 5 ppm was utilized along with a fragment mass tolerance of 0.02 Da. Up to 4
dynamic modifications were allowed per peptide. Dynamic modifications included deamidation
(0.984 Da, N, Q), oxidation (15.995 Da, M), GlyGly (114.043 Da, K), and LeuArgGlyGly
(383.228 Da, K) [14, 15]. For static modifications, all files were searched with carbamidomethyl
(57.021 Da, C) except enriched sample files that were searched with N-ethylamaleimide
(+125.0477, C).The Percolator node was used to calculate the false discovery rate (FDR) with a
strict cut off of 0.01 and validation based on q-value.
3 Results and Discussion
3.1 Epitope labeled lysozyme Western blot
In order to determine if the Boc-Gly-Gly-NHS
modification procedure of lysozyme was successful, a
Western blot utilizing the commercially available anti-
Gly-Gly was performed [3]. As is demonstrated in Fig.
2A, lysozyme and Boc-Gly-Gly-lysozyme was not
probed by the antibody due to the lack of available K-
ε-GlyGly groups. The epitope labeled protein was
identified by anti-Gly-Gly after Boc removal (Gly-Gly
lysozyme). Variable molecular weight shifts are
representative of different numbers of modified
lysines [3]. In order to further identify how many
lysines were modified and what peptides were being
modified, LC-MS/MS was performed on the epitope
labeled protein.
3.2 Protein standard characterization
Pure modified lysozyme was analyzed by LC-MS/MS
using the Thermo Q-Exactive High Field instrument. A
sequence coverage of 72.11% was achieved for the
protein, Fig. 2B. Of the 6 available lysines, 5 were
identified as having the Gly-Gly modification present.
Each Gly-Gly shifts the weight of a protein 114.04 Da;
therefore Gly-Gly modifications to 5 lysines within
lysozyme as well as the additional dynamic (O, D, L)
and static modifications (C) should create a shift of
954.171 Da for a total molecular weight of 17.184
kDa, Fig. 2A. These results suggested that not only
did the modification procedure successfully label
Fig. 2. (A) Western blot of Boc-Gly-Gly-
NHS modified lysozyme when probed
with anti-Gly-Gly. (B) The sequence for
lysozyme, but that almost all lysines had the Gly-Gly
modification.
Lysozyme (126608) and the identified
modified lysines. Identified peptides are
underlined.
3.3 Protein standard spike-in concentration
optimization
In order to determine the mass percent at which the
protein standard should be spiked into a complex sample, a range of concentrations of a 1:1
epitope modified to unmodified protein standard mixture was added to a complex M. oryzae

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background. Three concentrations, 0.1%, 1%, and 5% of the 1:1 spiked in control were added to
the extracted protein. Each sample was subsequently digested and analyzed by LC-MS/MS.
The modified protein standard was identified with a sequence coverage of 76.19%, 80.27%,
80.27%, in the 0.1%, 1%, and 5% spiked-in samples, respectively. Both Gly-Gly modified lysine
Fig. 3. Protein standard concentration optimization. The peptide ckGTDVQAWIR identified in
each sample spiked with the protein standard at three different concentrations.
containing peptides (Fig. 3, Table 1, Supplemental Material) and unmodified lysine containing
peptides were identified in each tested concentration, suggesting that 0.1% concentrations of
the protein standard were capable of not only being identified but would provide information on
Gly-Gly containing peptides and resulting information about enrichment efficiency.
3.4 Gly-Gly enrichment efficiency examined
Application of the epitope modified protein standard was performed using the Cell Signaling
Enrichment kit. Using the optimized concentration of the protein standard mixture, 0.1% of the
protein standard mixture was combined with the M. oryzae complex background, and Gly-Gly
enrichment using anti-Gly-Gly was performed. The enriched peptides, the flow through, and a
non-enriched sample were analyzed by LC-MS/MS. Fig. 4A is representative of an enriched
modified peptide, ckGTDVQAWIR, identified in the enriched sample; the unmodified counterpart
of this peptide was identified in the non-enriched sample Fig. 4B. The percent coverage of the
protein standard was 78.91%, 10.88%, and 32.65%, respectively, with 100% (5/5) of identified

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protein standard peptides having the Gly-Gly modification in the enriched sample, no Gly-Gly
modified peptides in the flow through sample, and 25% (1/4) of peptides with the Gly-Gly
modification in the non-enriched sample (Table S2). In order to determine how well the
enrichment method was applied, enrichment efficiency was calculated using Equation 1:
Enrichment efficiency = (Ʃx’Enriched / (Ʃx’Enriched + Ʃx’Non-Enriched + ƩxEnriched + ƩxNon-Enriched))
Enrichment efficiency, derived from the sum of enriched modified protein peptides peak areas,
x', divided by the total of the sum of all enriched peptide peak areas and the sum of the total
non-enriched peptide peak areas [16], was calculated to be 99.3% (Table 1). By using the
spiked in protein standard, we were able to observe, with the use of a simple calculation, that
Fig. 4. (A) The Gly-Gly modified protein standard peptide ckGTDVQAWIR in the enriched
sample. (B) The unmodified peptide cKGTDVQAWIR in the non-enriched sample.
enrichment was performed as a result of the 99.3% enrichment of the protein standard.
4. Conclusions
Post-translational modification enrichment processes are at the forefront of the field of
proteomics, and the development of these methods allows researchers to continue to gather
valuable information on dynamic changes occurring throughout the proteome. This information

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is then pieced together enabling the creation of a more complete picture used to explain what
regulatory mechanisms are controlling responses to applied stimuli. However, with enrichment
methods often comes extensive sample handling and introduction of variability that is difficult to
account for. We demonstrate that with the use of a spiked in Gly-Gly epitope modified protein
standard, peptide peak area of modified peptides can be used to calculate enrichment
efficiency. Because the protein standard is spiked in from the beginning of sample handling and
is characteristic of a ubiquitinated protein, any introduced variability that results in a change to
the enrichment process and outcomes are revealed to the researcher.
Acknowledgements:
This work was supported by the United States Department of Agriculture (USDA-NIFA-AFRI-
003958).

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ACCEPTED MANUSCRIPT
Sequence
Modification
Enriched
Non Enriched
Peak Area Peak Area
cELAAAmk
C1(Nethylmaleimide); M7(Oxidation); K8(GlyGly)
C1(Nethylmaleimide); K8(GlyGly)
9.67E+08
9.59E+08
1.26E+10
1.89E+10
1.65E+10
2.37E+08
0.00E+00
1.39E+07
1.08E+09
0.00E+00
0.00E+00
0.00E+00
1.42E+07
7.62E+06
0.00E+00
0.00E+00
5.18E+07
0.00E+00
0.00E+00
0.00E+00
cELAAAMk
cELAAAmkR
C1(Nethylmaleimide); M7(Oxidation); K8(GlyGly)
C1(Nethylmaleimide); K8(GlyGly)
cELAAAMKR
ckGTDVQAWIR
ckGTDVqAWIR
cKGTDVQAWIR
ckGTDVQAWIRGcRL
GYSLGNWVcAAkFESNFNTQATNR
C1(Nethylmaleimide); K2(GlyGly)
C1(Nethylmaleimide); K2(GlyGly); Q7(Deamidated)
C1(Nethylmaleimide)
C1(Nethylmaleimide); K2(GlyGly); C13(Nethylmaleimide)
C9(Nethylmaleimide); K12(GlyGly)
GYSLGNWVcAAkFESNFNTQATNRN
TDGSTDYGILQINSR
C9(Nethylmaleimide); K12(GlyGly)
HGLDNYRGYSLGNWVcAAkFESNFN
TQATNR
C16(Nethylmaleimide); K19(GlyGly)
1.75E+08
0.00E+00
kIVSDGNGmNAWVAWR
K1(GlyGly); M9(Oxidation)
K1(GlyGly); N7(Deamidated); M9(Oxidation)
K1(GlyGly)
2.08E+08
3.62E+07
1.72E+08
2.44E+07
0.00E+00
0.00E+00
0.00E+00
0.00E+00
kIVSDGNGmnAWVAWR
kIVSDGNGMNAWVAWR
NLcNIPCSALLSSDITASVNCAKk
C3(Nethylmaleimide); C7(Nethylmaleimide);
C21(Nethylmaleimide); K24(GlyGly)
C3(Nethylmaleimide); K4(GlyGly)
NRckGTDVQAWIR
NRcKGTDVQAWIR
NTDGSTDYGILQINSR
VFGRcELAAAmkR
WWcNDGR
1.49E+08
5.56E+06
5.28E+07
4.98E+07
0.00E+00
0.00E+00
0.00E+00
0.00E+00
1.39E+08
0.00E+00
2.51E+07
7.28E+07
C3(Nethylmaleimide)
C5(Nethylmaleimide); M11(Oxidation); K12(GlyGly)
C3(Nethylmaleimide)
WWcNDGRTPGSR
C3(Nethylmaleimide)
Table 1. Protein Standard Enrichment Analysis
Note: Please refer to supplementary material for peptide sequence data.