Agilent AssayMAP Bravo Technology
Enables Reproducible Automated
Phosphopeptide Enrichmentfrom Complex Mixtures Using
High‑Capacity Fe(III)‑NTA Cartridges
Application Note
Automated Peptide Sample Preparation for LC/MS

Authors

Abstract

Jason D. Russell and Steve Murphy

Immobilized metal affinity chromatography (IMAC) using a nitrilotriacetic

Agilent Technologies, Inc.

acid (NTA) chelating ligand functionalized with Fe(III) is regarded as one

Madison, WI, USAof the most selective phosphopeptide enrichment techniques for LC/MS
applications. However, the use of IMAC for phosphopeptide enrichment often
results in unacceptably high sample‑to‑sample variability. Phosphopeptide
enrichment is reliant on manual sample preparation methods resulting in
workflows lacking robustness except in the hands of highly trained scientists.
Recently, several protein and peptide sample preparation workflows wereautomated using the Agilent AssayMAP Bravo platform, which reduced sample
preparation variability, provided a clear path for sample processing scalability, andenabled customized workflow development without the needfor in‑house automation expertise. We now report on the use of high‑capacity
Agilent AssayMAP Fe(III)‑NTA cartridges for automated phosphopeptide
enrichment featuring the AssayMAP Bravo. We describe the capacity of
AssayMAP Fe(III)‑NTA cartridges, explain chemistry considerations for successful
phosphopeptide enrichment by IMAC, and demonstrate that thousands ofphosphopeptides can be reproducibly purified from yeast tryptic digests with highspecificity in an automated fashion facilitated by high‑resolution LC/MS.

Introduction
Reversible protein phosphorylation is
the primary regulator of intracellularsignaling. Understanding signaltransduction with protein‑level resolution
is critical for characterizing abnormalities insignal transduction pathways regulated
by protein phosphorylation. The studyof global protein phosphorylation
has been revolutionized by theuse of liquid chromatography andhigh‑resolution mass spectrometry(LC/MS) whereby phosphopeptides
generated by enzymatic proteolysisof phosphoproteins are used for bothcharacterization and quantification ofphosphoproteins and phosphorylation
dynamics. Phosphopeptide enrichment isrequired to achieve the most sensitive
analysis of the phosphoproteome by LC/
MS and one leading approach forphosphopeptide enrichment is based
upon the use of immobilized metal affinity
chromatography (IMAC). IMAC can beused to selectively bind phosphopeptides
from complex protein digests, particularly
when Fe(III) is immobilized onto resinfunctionalized with the tetradentate
metal‑chelating ligand nitrilotriacetic acid
(NTA). Despite the widespread use of
IMAC for phosphopeptide enrichment,
there is no clear literature consensus on
sample and reagent chemistries that
reproducibly yield high phosphopeptide
recovery and selectivity1‑24.
Global phosphopeptide enrichment for
LC/MS is one of the more challenging
sample preparation workflows to perform
reproducibly. Perhaps, the greatest
obstacle to reproducible phosphopeptide
enrichment by IMAC is optimizationof the chemistry conditions such thatthey are sufficiently stringent to allow
selective binding of phosphopeptides
without compromising enrichment
reproducibility or phosphopeptide
yield. Less than optimal conditions
may promote low phosphopeptide
selectivity through copurification ofacidic, unmodified (not phosphorylated)
peptides. In contrast, conditions canbe overly stringent, leading to poor orinconsistent recovery of phosphopeptides.
Solution chemistry is a critical factor

governing success for phosphopeptide
enrichment using IMAC. Samples forphosphopeptide enrichment using theFe(III)‑NTA IMAC strategy almost always
require a cleanup (desalting) step toremove sample components that may
interfere with enrichment. Desalted
samples are typically dissolved in aphosphate‑free, TFA‑containing, low‑pH
solution (~ pH 2.4 to 2.8) with a large
percentage of organic solvent. Low‑pH
conditions reduce nonspecific bindingby facilitating protonation of acidicside‑chains and C‑termini of unmodified
peptides, whereas the phosphoryl
groups of phosphopeptides remain
negatively charged to promote binding toimmobilized Fe(III) through a combinationof electrostatic interactions and metal
coordination25,26. The presence of organic
solvent in the sample and wash solutions
is also essential to minimize nonspecificbinding. Organic solvent modulates thedegree of ionization of acids and bases
and the magnitude of the change in the
degree of ionization can vary dramaticallydepending on the chemical species20, 27‑30.
Achieving high phosphopeptide selectivity
requires determining conditions thatmaximize the difference in the ionization
states of phosphoryl and carboxyl groups
such that under a given set of solvent
conditions, phosphoryl groups arenegatively charged, while carboxyl groups
are neutral to promote selective bindingof phosphopeptides to immobilized
metal cations16, 20. Once enrichment
conditions have been optimized, thetask of scaling sample preparation andtransferring methodology to achieve
sample‑to‑sample and site‑to‑site
reproducibility is daunting considering themanual nature of many phosphopeptide
enrichment workflows.
Several core yeast protein sample
preparation tasks have been automatedusing the Agilent AssayMAP Bravo
sample preparation platform (for example,
immunopurification, protein digestion,
peptide cleanup, and so forth) throughthe use of microchromatography
cartridges and flexible and task‑centricautomation protocols. With thesame automation platform, we now
demonstrate reproducible and selective
phosphopeptide enrichment by IMAC
2

using high-capacity AssayMAP
Fe(III)‑NTA cartridges. Each AssayMAP
Fe(III)-NTA cartridge contains a 5-μL
packed resin bed containing NTA
resin precharged with more than
100 nmol of Fe(III) per cartridge. Whenused in tandem with the AssayMAP
Phosphopeptide Enrichment v2.0 App,thousands of phosphopeptides can be
reproducibly enriched from complex
protein digests. Here, we detail thecapacity of AssayMAP Fe(III)-NTA
cartridges, systematically evaluate thechemistry considerations for successful
phosphopeptide enrichment by IMAC,
and demonstrate that thousands ofphosphopeptides can be reproducibly
purified from complex mixtures withhigh specificity in an automated fashion
enabled by the AssayMAP Bravo andhigh‑resolution LC/MS.

Experimental
Bulk in-solution digestion andpeptide cleanup

Bovine a-casein (Sigma-Aldrich)
was diluted to 30 μg/μL in water.
A denaturation solution containing
9 M urea, 125 mM Tris (pH 8.0), and12.5 mM TCEP was added at fourtimes the sample volume. Samples
were denatured, and reduced at
55 °C for 1 hour with frequent mixing.
Iodoacetamide was added to a final
concentration of 20 mM, and was
incubated at room temperature in thedark for 40 minutes. Samples were diluted
with 50 mM Tris (pH 8.0) to reduce theconcentration of urea to 1.25 M. Trypsin
was added to yield an enzyme:protein
ratio of 1:30. Samples were incubatedat 37 °C for 2–4 hours, after which an
additional dose of trypsin was added toyield a final ratio of 1:20. Samples wereincubated overnight at 37 °C.
Yeast (Saccharomyces cerevisiae)
protein extracts from whole-cell lysates
were provided by Blue Sky BioServices(Worchester, MA). Lysates were preparedusing denaturing lysis conditions adaptedfrom Swaney et al. and contained bothprotease and phosphatase inhibitor
cocktails31. Clarified samples (5 mg/mL)
were denatured and reduced by adding

an equal volume of a denaturation
mixture containing 8 M urea, 100 mM
Tris (pH 8.0), and 10 mM TCEP, followed
by incubation at 55 °C for 1 hour withfrequent mixing. Iodoacetamide wasadded to a final concentration of 15 mM,
and samples were incubated at room
temperature in the dark for 40 minutes.
Samples were diluted with 50 mM Tris
(pH 8.0) to reduce the urea concentrationto < 1.25 M, and trypsin was added at
an enzyme:protein ratio of 1:40. Samples
were incubated at 37 °C for 2 hours,
after which an additional dose of trypsin
was added to yield a final ratio of 1:20.
Samples were incubated overnight at
37 °C.
Digestion was quenched by addition of
TFA to a final concentration of 1 % toreduce the pH to ~2.5. Samples weredesalted using Agilent Bond Elut C18
cartridges with a bed mass at least 30x
larger than the sample mass. Desalted
digests were aliquoted as 5 mg (a-casein)or 1.5 mg (yeast) quantities, lyophilized,
and stored at –80 °C.

Phosphopeptide enrichment using
Agilent AssayMAP Fe(III)-NTA
cartridges

The Agilent AssayMAP Phosphopeptide
Enrichment v2.0 App, included with theAgilent AssayMAP Bravo Protein Sample
Prep Workbench v2.0 software suite,
was used for automated phosphopeptide
enrichment experiments with AssayMAP
Fe(III)-NTA cartridges. Chemistry
conditions were systematically evaluated,
and the effects of these conditions
on phosphopeptide enrichment are
explained in the Results and Discussionsection. See Table 1 for representative
on‑deck samples and reagents used forphosphopeptide enrichment. Trypticallydigested, desalted, and lyophilized
samples in 2-mL tubes were diluted to
0.5–5.0 μg/μL with 80 % acetonitrile(ACN)/0.1 % TFA. The aqueouscomponent of this solution was first
added to the samples, samples werevortexed, and then ACN was added.
The samples were further vortexed until
visible particulates were dissolved. The
samples were centrifuged at 6,000 × g
for 5 minutes to pellet any remaining

Table 1. Representative samples and reagents used for phosphopeptide enrichment with theAgilent AssayMAP Phosphopeptide Enrichment v2.0 App.
Representative on-deck samples and reagents
Agilent AssayMAP Bravo App - Phosphopeptide Enrichment v2.0
Sample: digested, desalted, and lyophilized

S. cerevisiae protein lysate or bovine a-casein diluted to
0.5–5.0 µg/µL in 80 % ACN/0.1 % TFA

Priming buffer

50 % ACN/0.1 % TFA

Equilibration and internal cartridge wash buffer

80 % ACN/0.1 % TFA

Elution buffer

1 % Aqueous ammonia (ammonium hydroxide), pH ~11

Wash station

Dieonized water

Note: All buffers were made as v/v mixtures3

microparticulates. The samples weremanually transferred from tubes into
polypropylene 96-well plates (either
PCR or V-bottom plates). A solution of
50 % ACN/0.1 % TFA was used as thepriming buffer, 80 % ACN/0.1 % TFA
was used as the equilibration andinternal cartridge wash buffer, and 1 %aqueous ammonia (commonly referred
to as ammonium hydroxide, pH ~11)
was used as the elution buffer. Default
protocol values, with the exception of
the number of columns of cartridges
and sample volume, were used forphosphopeptide enrichment of a-casein
unless otherwise indicated. Refer toFigure 1 for the representative protocol
settings and labware selections used
for phosphopeptide enrichment fromyeast. Samples were directly eluted from
AssayMAP Fe(III)-NTA cartridges into a
PCR plate containing 2.5 μL neat formic
acid for immediate sample acidification.
The concentrations of sample eluateswere adjusted with 0.1% formic acid,and sample plates were sealed with an
Agilent PlateLoc Thermal Microplate
Sealer using pierceable film, and stored in
an autosampler at 4 °C.

Figure 1. Agilent AssayMAP Phosphopeptide Enrichment v2.0 App. Representative applications settings and labware selections for phosphopeptide
enrichment from yeast digests. Settings and labware were adjusted based upon the needs of the particular experiment.

LC/MS and data analysis

LC/MS analyses were conducted onan Agilent 6550 iFunnel Q-TOF LC/MS
system equipped with a Dual Agilent Jet
Stream ESI source (Table 2). The mass
spectrometer was coupled to an Agilent
1290 Infinity Binary LC system housing
an AdvanceBio Peptide Mapping C18
column (2.1 × 150 or 250 mm, 2.7 µm,
120 Å pore size) maintained at 55 °C.
Mobile phase A was 0.1 % formic acid inwater, and mobile phase B was 99.9 %ACN with 0.1 % formic acid. A flow rateof 400 µL/min was used for gradientelution. The autosampler was configured
for 96-well PCR plates, and injection
volumes were varied based upon theparticular experiment. Auto MS/MS
mode was performed and the 10–15 mostabundant precursors were selected for
MS/MS per cycle using narrow isolation
and an active exclusion time of 1 minute.
The acquisition scan rate was set at
8 Hz for MS and 3.5 Hz for MS/MS.
Peak area analyses were performed
using the MS1 Full-Scan Filtering
option in Skyline (64-bit, version 3.1),
and Spectrum Mill (Rev B.04.01) wasused for peptide identification. MS/MS

spectra were searched against a custom
database containing protein sequences
for bovine a-s1 and a-s2 or a protein
database constructed from the UniProt
reference proteome for Saccharomyces
cerevisiae (strain ATCC 204508/S288C).
Trypsin specificity was selected, and up
to three missed cleavages were allowed.

Carbamidomethylation was searched
as a fixed modification while oxidized
methionine and phosphorylated serine,
threonine, and tyrosine were searched as
variable modifications. Peptide-spectrum
matches were filtered to ~1.2 % false
discovery rate using the default auto
threshold values.

Table 2. Instrumentation and consumables.
Agilent AssayMAP Bravo Cartridges, analytical instrumentation, and software
Automation
Core automation platform

Agilent AssayMAP Bravo (G5542BA)

Agilent AssayMAP cartridges(96 cartridges/rack)

96 Fe(III)-NTA cartridge rack (G5496-60085)
96 Resin-free cartridge rack (G5496-60009)

Additional accessories

Agilent PlateLoc Thermal Microplate Sealer (G5402A)

LC/MS
Mass spectrometer

Agilent 6550 iFunnel Q-TOF LC/MS
Dual Agilent Jet Stream ESI

LC systems and columns

Agilent 1290 Infinity LC System
Agilent AdvanceBio Peptide Mapping Column (C18):
Anayltical: 2.1 × 250 mm, 2.7 µm (651750-902)
2.1 × 150 mm, 2.7 µm (653750-902)
Guard:
2.1 mm Fast Guard (851725-911)

SoftwareAgilent AssayMAP Protein Sample Prep Workbench v2.0
Agilent MassHunter Acquisition for TOF/Q-TOF, Rev. B.06.01
Spectrum Mill MS Proteomics Workbench, Rev. B.04.01
Skyline (64-bit), version 3.1

4

With AssayMAP cartridges, boundanalytes can be eluted from cartridgesin ≤ 20 μL if a sufficiently strong elution
buffer is used. Small-volume elution isenabled by the ability to strictly control
the elution flow rate unidirectionallythrough the 5-μL packed beds ofAssayMAP cartridges. Analyte elution
conducted in this format maximizes
stationary phase pore penetration by
the elution buffer, and minimizes thevolume required for complete elution. To
demonstrate small-volume elution using
Fe(III)-NTA cartridges, 50 or 150 μg of
a-casein digest was bound and eluted
from cartridges using 1 % aqueous
ammonia.

TIC Counts

×10 7
2

phosphopeptide signal was measured in
4 μL of eluate (less than one cartridge
bed volume) with 99.9 % of the total
phosphopeptide signal measured in thefirst 10 μL of eluate collected from the
cartridge.

Sample input

1
0

TIC Counts

Bovine a-casein is a well-characterized
phosphoprotein often used to evaluate
the efficacy of phosphopeptide
enrichment strategies. Commercial
preparations of bovine a-casein arecommonly mixtures of two forms, a-s1
and a-s2, containing 8–13 phosphates permolecule mapping to more than 20 known
sites of phosphorylation32,33. In preliminary
experiments, 31 phosphopeptides wereidentified from a-casein tryptic digests,
and of those phosphopeptides, 16 were
fully tryptic, 12 contained one missedcleavage, and three contained two missedcleavages. Twenty-four phosphopeptides
were singly phosphorylated, four weredoubly phosphorylated, and three weretriply phosphorylated. Phosphopeptides
from 150 μg of desalted a-casein tryptic
digest were enriched using AssayMAP
Fe(III)-NTA cartridges. The sample
input, the cartridge flow through fromsample loading, and the cartridge eluate
were analyzed, and the MS total ion
chromatogram (TIC) of each is displayedin Figure 2. More than 92 % of thepeptide TIC in the eluate originated fromthe 31 phosphopeptides identified froma-casein, demonstrating very selectiveenrichment from this model system.

Cartridge flow through2
1
0

TIC Counts

Phosphopeptide enrichment frombovine a-casein

A custom automation protocol wasused to collect 2-μL fractions of eluate.
The total phosphopeptide peak area(summation of the 31 phosphopeptide
EIC peak areas) was plotted against
the cumulative elution volume(Figure 3). More than 99 % of the total

Cartridge eluate

2

*
*

1
0

1

**
** *
2

3

**
* *
* *
* ** **** * * * * ** *
4
7
5
6
8
9
10
Acquisition time (min)= Phosphopeptide
11

12

13

14

15

Figure 2. High-selectivity phosphopeptide enrichment. Phosphopeptides were enriched from 150 μgof trypsinized and desalted a-casein using Fe(III)-NTA cartridges. The MS TICs from the sample input(black trace), cartridge flow through (red trace), and cartridge eluate (green trace) are shown. More than
92 % of the total peptide TIC signal in the cartridge eluate was from a-casein phosphopeptides.

100

Load mass
50 µg% of Total phosphopeptide signal

Results and Discussion

100 µg

80
60
40
20
0

2

4

6
8
Cumulative elution volume (µL)

10

Figure 3. Small-volume elution. Fe(III)-NTA cartridges were loaded with 50 or 150 μg of desalted a-casein
tryptic digest and eluted in 2-μL fractions using 1 % aqueous ammonia (pH ~11). More than 99 % of thetotal phosphopeptide signal was concentrated in 4 μL of eluate (< 1 cartridge bed volume). Complete
elution was achieved with 10 μL of elution buffer.

5

of 31 phosphopeptides were summed,
and the average total phosphopeptide
signal from the replicates was plottedagainst the mass of sample loaded onto
cartridges (Figure 4). As an additional
point of reference, the phosphate content
of the input sample was estimatedusing reported literature values forbovine a-casein and plotted against themass of sample loaded32,33. The total
phosphopeptide signal was constant
up to 150 μg (~60 nmol of phosphate),
after which the signal began to decreaseas the cartridge became saturated withphosphopeptides and global binding
capacity was exceeded.

MS Signal (n = 4)
Estimated phosphate content in sample load

×10 8
2.0

120

1.5

100

1.0

80

0.5

40

0

25

50

75

100
150
200
250
300
Mass of a-casein tryptic digest (µg)

350

400

0

Estimated phosphate content (nmol)in the cartridge flow through suggested
that the global phosphopeptide binding
capacity of the Fe(III)-NTA cartridges
had been exceeded. To confirm this, acapacity study was performed in which
25–400 μg of a-casein digest was loaded
onto Fe(III)‑NTA cartridges (n = 4)
yielding 40 parallel phosphopeptide
enrichments. Cartridge eluates werediluted to 1 μg/μL based upon the massof sample loaded onto cartridges withequal volumes injected for each sample.
If enrichment was 100 % efficient at eachload mass, the total phosphopeptide
signal should remain constant acrossthe tested range. The EIC peak areas

Total phosphopeptide EIC peak area

In the experiments where 150 μgof a-casein digest was loaded
onto Fe(III)-NTA cartridges, there
was a small, but measureable,
phosphopeptide signal in the cartridge
flow through. By comparison, there
was no phosphopeptide signal in theflow through from the 50 μg cartridgeload. The overwhelming majority ofthe phosphopeptide signal in the flow
through from 150 μg sample loads wasfrom highly basic phosphopeptides,
typically those phosphopeptides with one
or two missed cleavages. After ruling out
the sample-loading flow rate as the root
cause, the presence of phosphopeptides

Figure 4. Global phosphopeptide cartridge capacity with an a-casein model system. Digested anddesalted a-casein was loaded onto Fe(III)-NTA cartridges in amounts spanning 25 to 400 μg. The total
peak area signal generated from EICs of all identified phosphopeptides was plotted against the massof digest loaded onto cartridges. The estimated phosphate content of the sample was derived from the
known phosphate content of bovine a-casein. Above 150 μg of sample (~60 nmol of phosphate), thecartridge capacity was exceeded for global quantitative phosphopeptide binding.

6Each of the 31 phosphopeptides
was examined in detail to better
understand phosphopeptide-specificdifferences in binding behavior. In
Figure 5, the signals of two fully tryptic
phosphopeptides were plotted alongwith missed-cleavage versions of those
peptides to demonstrate how binding
capacity is affected by phosphopeptide
sequence. The peak area of the highly
acidic phosphopeptide EQLsTSEENSK
was constant up to 400 μg, whereas
the peak area of the phosphopeptide
EQLsTSEESKK, containing a single missedcleavage, began to decrease beyond
the 250 μg load amount primarily due
to the peptide’s increased basicity. The
addition of a second phosphoryl group to

EQLsTSEENSK

B

×10 7
5.0

Phosphopeptide
EIC peak area

×10 5
1.0

Phosphopeptide
EIC peak areaA

the missed-cleavage phosphopeptide to
produce EQLsTsEESKK resulted in stablesignal up to 400 μg. To further illustrate
the selectivity IMAC enrichment has foracidic phosphopeptides, the peak areasof three peptides containing zero, one,or two missed cleavages were tracked(Figure 5). The signal for the fully tryptic
phosphopeptide VPQLEIVPNsAEER began
to decrease at the 250 μg load mass.
The addition of one or two C-terminal
lysines caused by enzymatic-missedcleavage reduced the cartridge capacity
for this phosphopeptide to 150 μg
and 75 μg sample loads, respectively.
Because electrostatic interaction plays
such a dominant role in binding, acidic
phosphopeptides are able to competitively

0.5

0

0

Phosphopeptide
EIC peak area

YKVPQLEIVPNsAEER

3

0
×10 6
2

EQLsTsEENSKK
Phosphopeptide
EIC peak area

×10 6

3

0

2.5

×10 7
6

EQLsTSEENSKK

0.66

VPQLEIVPNsAEER

0
6

Phosphopeptide
EIC peak area

Phosphopeptide
EIC peak area

×10
1.2

displace basic phosphopeptides from the
cartridge resin bed during the sample
loading process. The fact that phosphoryl
moieties bind to immobilized Fe(III)through a combination of electrostaticinteractions and metal coordinationmeans that to minimize bias one must
work within the global phosphopeptide
binding capacity25. Because automatedphosphopeptide enrichment isreproducible with the AssayMAP Bravo,
any phosphopeptide-specific biases canbe accurately measured and addressed
through similar cartridge capacity studies,
as is later demonstrated with yeast
lysates.

1

0

25
50
75
100 150 200 250 300 350 400
Mass of a-casein tryptic digest loaded onto cartridges (n = 4)KYKVPQLEIVPNsAEER

25
50
75
100 150 200 250 300 350 400
Mass of a-casein tryptic digest loaded onto cartridges (n = 4)

Figure 5. Sequence-dependent phosphopeptide capacity. The individual phosphopeptide capacity of an Fe(III)-NTA cartridge is dependent on
the phosphopeptide sequence and total mass of digest loaded onto the cartridge. (A) Cartridge capacity for the fully tryptic phosphopeptide
EQLsTSEENSK decreases with the addition of lysine as a result of enzymatic missed cleavage. Capacity for the missed-cleavage peptide is increased
upon the addition of second phosphoryl group. (B) Cartridge capacity incrementally decreases for the fully tryptic phosphopeptide VPQLEIVPNsAEER
as two additional lysines are added as a result of enzymatic missed cleavage. As the sample mass load increases above ~150 μg, acidic
phosphopeptides competitively displace relatively basic phosphopeptides from the cartridge resin bed.

7

aspartic acid, or phosphoric acid, thedegree of ionization decreases forglutamic and aspartic acid while thedegree of ionization of phosphoric acidis relatively constant.20 This suggeststhat as the % ACN increases in asample containing an acid modifier(such as TFA or acetic acid), carboxylgroups of peptides become protonated(neutral) while the phosphoryl groupsof phosphopeptides remain negatively
charged. This permits selective binding of
the negatively charged phosphoryl groupof phosphopeptides to the positively
charged iron immobilized on the NTA
resin.

Phosphopeptide selectivity
Phosphopeptide identifications
100

3,000

1.00 %

80

2,500
2,000

60

1,500
40
1,000
20

500
0

0.01 %
0.10 %% Phosphopeptide selectivity

We next evaluated the chemistry andprotocol factors affecting phosphopeptide
enrichment from yeast, a widely usedmodel system in the study of signaltransduction pathways controlled byphosphorylation. Complex peptide
mixtures were generated by bulk tryptic
digestion and reversed-phase desaltingof protein extracts from yeast wholecell lysate. Sample solutions and wash
buffers comprising 0.01, 0.1, or 1.0 % TFA,
or acetic acid in 80 % ACN were used toenrich phosphopeptides from 500 μg ofyeast digest. Samples were loaded onto
cartridges (n = 3) and washed with abuffer containing the same acid type andconcentration as the samples. As shownin Figure 6, at 0.01 % and 0.1 % both TFA
and acetic acid generated very similar
numbers of distinct phosphopeptide
identifications (~2,300). However, at 0.1 %the average phosphopeptide selectivity
increased from 80 % to 96 % for TFA,
and from 82 % to 91 % for acetic acid.
As the acid percentage was increased
to 1.0 %, the number of phosphopeptide
identifications dramatically decreased
with TFA from 2,375 to 550, accompanied
by a decrease in selectivity to 87 %.
With 1.0 % acetic acid, a more
moderate decrease in phosphopeptide
identifications occurred (2,266 to 1,783),
but phosphopeptide selectivity improved
to 97 %. Overall, the use of 0.1 %TFA produced greatest number ofidentifications while maintaining highselectivity, although the use of 0.1 %acetic acid produced comparable results.

Control of sample and wash solution
pH is imperative to achieving efficientand reproducible phosphopeptide
enrichment. One of the keys to selectively
enriching phosphopeptides is exploiting
the differences in ionizability of thephosphoryl group of phosphopeptides
and carboxyl groups of unmodified
peptides (while also considering theionizability of the acid modifier). At a
fixed concentration of acid modifier, theionizability of acidic and basic analytes
can be effectively tuned by the additionof organic solvent28-30. It has beenshown that as acetonitrile is titrated
into solutions containing glutamic acid,

Distinct phosphopeptides

Phosphopeptide enrichment fromyeast whole-cell lysate

Acetic acidTFA

0

Figure 6. Effect of acid type and concentration on phosphopeptide enrichment. Digested and desalted
yeast digests (500 μg) were loaded onto Fe(III)-NTA cartridges with varying amounts of acetic acid or TFA
in the presence of 80 % ACN (n = 3). An acid concentration of 0.1 % yields the best balance of distinct
phosphopeptide identifications and phosphopeptide selectivity for both acids.

8

A

90

Distinct
phosphopeptides
1,600
1,800

% ACN in wash buffer

2,000
2,200

80

2,400
2,600

70

60

60

70

% ACN in sample

80

90

90

B

Phosphopeptide% selectivity
90
92
94

% ACN in wash buffer

To examine the effects of the % ACN on
phosphopeptide enrichment from yeast,
samples and wash solutions were madein 60, 70, 80, or 90 % ACN with 0.1 %TFA. Phosphopeptides from 500 μg ofyeast digest were enriched in triplicate
for each combination of sample and wash
buffer (16 total combinations). Heat maps
visualizing both the number of distinct
phosphopeptide identifications and% phosphopeptide selectivity are shownin Figure 7. The greatest yield of distinct
phosphopeptide identifications occurredwith 80 % ACN in both the sample andwash solutions. The heat map model
extrapolation suggests that a range from
~70–85 % ACN in the sample and wash
solutions would produce comparable
numbers of distinct phosphopeptide
identifications. There is a steep decreasein phosphopeptide identifications as the% ACN in the sample is reduced below
70 %. The condition where both thesample and wash solutions contained
60 % ACN produced only 55 % of theidentifications relative to the condition
with 80 % ACN in both solutions. In
terms of phosphopeptide selectivity,
all conditions yielded phosphopeptide
selectivity of at least 89 %. A broad rangeof conditions exist that will increase
the selectivity above 94 %, but the mostfavorable conditions include sample
solution with ~70–80 % ACN and awash solution with ≥ 70 % ACN. As thepercentage of ACN in the sample solution
is titrated below 70 % in the presence of
0.1 % TFA, it is likely that the phosphoryl
groups of phosphopeptides become
protonated reducing the overall numberof phosphopeptides that are captured andsubsequently identified. Such conditions
may favor high phosphopeptide
selectivity, but at the cost of fewer
distinct phosphopeptide identifications.

96

80

98
100

70

60

60

70

% ACN in sample

80

90

Figure 7. The effect of % ACN in the sample and wash buffers on phosphopeptide identifications (A)and phosphopeptide selectivity (B) from yeast digests (500 μg). With 0.1 % TFA, the greatest number of
peptide identifications and highest phosphopeptide selectivity is achieved with ~ 70–80 % ACN.

9

Because phosphopeptide enrichment
performance using AssayMAP Fe(III)‑NTA
cartridges is dependent on the % ACN
in the sample and wash solutions,evaporation from labware on the deckof the AssayMAP Bravo should beclosely monitored during the method
development process to evaluate if
evaporation is affecting phosphopeptide
enrichment. If it is determined that
evaporation is negatively affecting
enrichment, several steps can be taken to
mitigate evaporation:•

Adjust the sample volume and flowrate to reduce the total protocol
time (sample loading is the
typically the lengthiest process).

•

Adjust the sample and wash
buffer formulations to account forevaporation.

•

Use labware that minimizes
evaporation (avoid using open
reservoirs).

•

Use a larger volume labware
overage (see the Phosphopeptide
Enrichment v2.0 User Guide).

•

Run the protocol in a step-wisemanner such that labware isplaced on deck only when needed.

A contributor to poor phosphopeptide
selectivity using Fe(III)-NTA AssayMAP
cartridges is incomplete sample
solubilization. Often, samples forphosphopeptides enrichment have firstbeen desalted and lyophilized leaving
a dry or semidry solid. When an acidic
solution containing a high percentage
of ACN is added to dried samples,it can be challenging to completely
solubilize samples such that no
peptide microparticulates are present.
Microparticulates, to a large degree, arerepresentative of the peptide profile ofthe entire sample and contain mostly
unmodified peptides. Microparticulates
can reduce phosphopeptide selectivity
using AssayMAP Fe(III)-NTA cartridges
as illustrated in Figure 8 (top). Sample
microparticulates, which may only
represent a small fraction of sample
relative to the solubilized sample, canbe aspirated into the syringes fromsample wells. Particulate-laden sample
is then dispensed through an Fe(III)‑NTA
cartridge where microparticulates
accumulate at the top of the resin bed.
Performing an internal cartridge washwith high‑organic, acidic solutions
is inadequate to solubilize thesemicroparticulates, but such wash solution
compositions are essential for reducing
nonspecific binding and retaining
phosphopeptides. Phosphopeptide
elution employs aqueous solutions atbasic pH levels which solubilize thepeptide microparticulates at the top
the resin bed. The newly solubilized
unmodified peptides coelute withbound phosphopeptides. The net
result is that phosphopeptide bindingis largely not affected by the presenceof microparticulates, but selectivity
decreases due to an increase inunmodified peptides in the eluate
originating from microparticulates.
To further illustrate this point, threedifferent strategies for sample
solubilization and particulateremoval were used prior to enriching
phosphopeptides from 500 μg of yeast
digest:1.

The aqueous component of 80 %ACN/0.1 % TFA was added tosamples followed by the addition of
the organic component. The samples
were vortexed, and suspended
microparticulates were pelleted bycentrifugation.

2.

A solution of 80 % ACN/0.1 % TFA
was added to samples. The samples
were vortexed, and suspended
microparticles were pelleted bycentrifugation.

3.

A solution of 80 % ACN/0.1 % TFA
was added to samples. The samples
were vortexed, and no attempt
was made to remove suspended
microparticles.

As shown in Figure 8 (bottom),phosphopeptide selectivity decreased
from 96 % to 78 % correlating with thetendency to form and retain suspended
sample microparticulates due to thesample handling strategy. The number of10

distinct phosphopeptides identifications
was less affected by the presence of
microparticulates because the vast
majority of the sample was solubilized ineach condition. Phosphopeptide binding
capacity is not altered by the presenceof microparticulates, and the level ofcontamination from unmodified peptides
was not high enough to severely restrict
phosphopeptide sampling during Auto
MS/MS. However, a reduction in both
phosphopeptide selectivity and number ofidentifications will be exacerbated withincreased levels of microparticulates.
For best enrichment performance, only
particulate-free samples should be used.
A critical parameter for all sample
preparation protocols using AssayMAP
cartridges is the flow rate at whichsamples are loaded onto cartridges.
A key to successful analyte binding,assuming the chemistry conditions
have been optimized, is providing
analytes sufficient time to penetrate
the pores of resin and interact withthe stationary phase. Yeast digest(500 μg) was loaded onto Fe(III)-NTA
cartridges in four separate experiments
using a sample-loading flow rate of 2.0,
3.5, 5.0, or 6.5 μL/min (n = 3) and thedistinct number of phosphopeptides
and phosphopeptide selectivity wereassessed (Figure 9). The 2.0 and
3.5 μL/min sample‑loading flow rates
produced nearly identical results. As the
sample-loading flow rate was increased
beyond 3.5 μL/min, both the number ofdistinct phosphopeptide identifications
and phosphopeptide selectivity declined.
Relative to 6.5 μL/min, the 3.5 μL/minflow rate produced 9.4 % more distinct
phosphopeptide identifications, but
required nearly twice as much time
to load the sample. The decrease in
selectivity with increasing flow rate
was a direct result of capturing fewer
phosphopeptides as the number of
unmodified peptides identified at eachflow rate was relatively constant.
Ideally, the sample-loading flow rateshould be optimized for each set ofsample conditions; however, using
Phosphopeptide Enrichment v2.0 App’sdefault sample loading flow rate of
5 μL/min will likely be very close to the
optimum flow rate for many experimental
conditions.

Phosphopeptide selectivity
Phosphopeptide identifications

Distinct phosphopeptides

80

2,500
2,000

60

1,500
40
1,000
20

500
A

B

C

0

D

Phosphopeptide selectivity
Phosphopeptide identifications

80

3,000
60

2,500
2,000

40

1,500
20

1,000
500

1
2
3
Solubilization technique% Phosphopeptide selectivity

Distinct phosphopeptides

3,500

0

Figure 9. Effect of sample-loading flow rate on phosphopeptide enrichment.
Yeast digests (500 μg) were loaded onto Fe(III)-NTA cartridges at four
different sample-loading flow rates to determine the effect of flow rate on
phosphopeptide identifications and selectivity (n = 3). With a sample‑loading
flow rate above 3.5 μL/min, both the number of phosphopeptide
identifications and phosphopeptide selectivity decrease indicating
insufficient residence time for phosphopeptide binding.

100

4,000

2.0
3.5
5.0
6.5
Sample loading flow rate (µL/min)% Phosphopeptide selectivity

100

3,000

0

Figure 8. The effect of sample particulates on phosphopeptide enrichmentfrom yeast. (Top) Sample particulates containing mostly unmodified peptides
can cause poor phosphopeptide selectivity with Fe(III)-NTA cartridges.
Sample containing particulates can be aspirated from sample wells (A); as
the sample is dispensed through cartridges, particulates will accumulate at
the top of the cartridge resin bed (B); particulates can remain at the top of
the resin bed after internal cartridge washing (C); particulates can solubilize
in elution buffer resulting in a large number of unmodified peptides coeluting
with phosphopeptides bound to the cartridge (D). (Bottom) How samples aresolubilized can dramatically alter phosphopeptide selectivity. Good sample
solubilization techniques (1) results in high phosphopeptide selectivity,
whereas poor solubilization techniques (3) result in reduced selectivity.
See text for details.

11marginally increased the number ofdistinct phosphopeptide identifications.
Despite loading 50 % more sample, the1,200 μg load mass resulted in only a
6.4 % increase in distinct phosphopeptide
identifications relative to the 800 μg
load mass. Average phosphopeptide
selectivity was greater than 94 % acrossthe tested range suggesting that excess
cartridge capacity does not negatively
affect selectivity. Performing a 2nd roundof enrichment provided very little benefit
below 800 μg as the average number ofdistinct phosphopeptides identified was
at least 100-fold less than the 1st roundof enrichment. At 800 μg and above, the
2nd round of enrichment produced an
appreciable number of phosphopeptide
identifications. At the 1,200 μg level,
greater than 50 % of the phosphopeptides

4,000

Phosphopeptide selectivity
1st Pass
2nd Pass

identified in the 2nd round of enrichment
were also identified in the 1st round ofenrichment. Phosphopeptides identified
only in the 2nd round of enrichment
tended to be more basic than those
peptides identified only in the 1st round ofenrichment. Phosphopeptides identified
only in the 2nd round of enrichment had an
average of 1.03 phosphorylated residues
per peptide, 2.33 basic residues (H, K, R),
and 2.49 acidic residues (D, E). By
comparison, phosphopeptides identified
only in the 1st round of enrichment had an
average of 1.25 phosphorylated residues,
1.55 basic residues, and 2.97 acidic
residues. These data corroborate thefindings with a-casein - as cartridge
capacity is exceeded, recovery of themost acidic phosphopeptides is favored.

Phosphopeptide identifications
1st Pass
2nd Pass

100
80

3,000
2,500

60

2,000
40

1,500
1,000

20

% Phosphopeptide selectivity

3,500
Distinct phosphopeptides

The analyte-dependent binding capacityof the Fe(III)-NTA cartridges was
described using digest of a-casein;
however, a similar experiment was
designed for the yeast model system
containing a more chemically diverse
population of phosphopeptides. This
experiment also differed in that the
cartridge flow through from the sample
loading and internal cartridge wash
steps were combined, lyophilized, andsubjected to an additional round ofphosphopeptide enrichment using new
Fe(III)-NTA cartridges. As the mass ofsample loaded onto cartridges increased
from 200 to 1,200 μg (n = 3 at each loadmass), the average number of distinct
phosphopeptide identifications also
increased (Figure 10). However, loading
additional sample above 800 μg only

500
0

200

400

600
800
1,000
Mass of yeast tryptic digest (µg)

1,200

0

Figure 10. Global phosphopeptide cartridge capacity with a yeast model system. Increasing amountsof yeast digest were loaded onto Fe(III)-NTA cartridges (1st pass) and the sample flow through was
subjected to a 2nd round of phosphopeptide enrichment with new cartridges (2nd pass). The number ofdistinct phosphopeptide identifications in the 1st pass began to plateau above the 800 μg load level,
whereas, the number of distinct phosphopeptide identifications steadily increased in the 2nd pass,suggesting that cartridge capacity had been exceeded.

12

As a final assessment of reproducibility,
500 μg of yeast digest was enrichedusing the optimized chemistry andsample-loading flow rate. In Figure 11,
the MS TICs of each of the four
technical replicates are shown revealingnearly identical profiles. On average,
2,515 distinct phosphopeptides wereidentified (CV = 4.7 %) with an average
phosphopeptide selectivity of 96.2 %(CV = 2.6 %). This represents a 60-foldincrease in phosphopeptide selectivity,and nearly 23 times more phosphopeptide
identifications relative to the control(unenriched) sample (Table 3).

×10 7
Replicate 1

1.50

Replicate 2
Replicate 3

TIC Counts

1.25

Replicate 4

1.00
0.75
0.50
0.25
0.00

5

10

15

20

25
30
35
Acquisition time (min)

40

45

50

55

Figure 11. Reproducible phosphopeptide enrichment from yeast. MS TIC overlay of four replicate phosphopeptide
enrichments from yeast digests (500 μg) with nearly identical profiles.

Table 3. Figures-of-merit for phosphopeptide enrichment replicates from yeast digests.
500 µgYeast enrichment

Distinct
peptidesDistinct
phosphopeptides

Phosphopeptide selectivity (%)

Control (5 µg)

7,097

111

1.6

Replicate 1

2,534

2,388

94.2

Replicate 2

2,543

2,494

98.1

Replicate 3

2,665

2,504

94.0

Replicate 4

2,711

2,674

98.6

Replicate average

2,613

2,515

96.2

Replicate CV

3.4 %

4.7 %

2.6 %

13

60

Conclusions

References

Phosphopeptide enrichment fromcomplex mixtures is one of the mostchallenging sample preparation tasks
to perform reproducibly for LC/MS. The
dependence on manual phosphopeptide
enrichment strategies makes protocol
reproducibility and scalability a significant
challenge. The Agilent AssayMAP
Bravo addresses these challenges by
providing an automation platform uponwhich phosphopeptide enrichment
can be performed reproducibly using
high-capacity AssayMAP Fe(III)-NTA
cartridges. Sample processing can bescaled to perform 1-to-96 enrichmentsin parallel, enabled by a dedicatedphosphopeptide enrichment protocol
included in the Agilent AssayMAP Protein
Sample Prep Workbench v2.0 software
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We have characterized the capacityof AssayMAP Fe(III)-NTA cartridges,
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15

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This information is subject to change without notice.
© Agilent Technologies, Inc., 2015, 2016
Published in the USA, March 16, 2016
5991-6073EN