GE Healthcare
Application note 28-4062-81 AA
2D-LC phosphopeptide
2D-LC analysis of phosphopeptides in brain tissue using
Ettan MDLC and Finnigan LTQ
Key words: phosphopeptide • two-dimensional
liquid chromatography (2D-LC) • Ettan MDLC •
LC-MS/MS • reversed-phase chromatography (RPC)
• strong cation exchange (SCX) chromatography •
linear ion trap mass spectrometer
Materials
NAP™ 10 Columns
17-0854-01
A 2D–LC-MS method was developed to analyze phosphopeptides in mouse brain tissue. The trypsin-digested tissue
was separated by strong cation exchange chromatography
(SCX), followed by reversed-phase chromatography (RPC)
using Ettan™ MDLC. The detection was performed by mass
spectrometry using neutral loss of phosphoric acid to
selectively detect the phosphorylated peptides. Several
phosphorylation sites were found, and a strategy for
confident assignment of these was developed.
PlusOne™ DTT
17-1318-01
PlusOne Tris
17-1321-01
Trypsin, sequencing grade
17-6002-75
Introduction
One of the most important post-translational modifications
is phosphorylation of serine, threonine, or tyrosine residues.
Phosphorylated proteins play important roles in a wide
range of biological processes, such as signal transduction,
apoptosis, and cell cycle control. Detection of phosphorylation sites by mass spectrometry in proteins extracted from
biological material is hampered by the low abundance, low
stoichiometry, and poor ionization of phosphopeptides (1).
In this work, a biocompatible nanoscale liquid chromatography (LC) system, Ettan MDLC, was used for separating
phosphopeptides. No metal ions that can chelate phosphate
groups are present in the fluid pathway of the LC system,
resulting in highly sensitive analyses (2).
Separation of the tryptic peptides was performed in two
dimensions, SCX followed by RPC. A Finnigan™ LTQ™
linear ion trap mass spectrometer was used for detecting
phosphopeptides by fragmenting all peptides that exhibited
a neutral loss of phosphoric acid.
Products used
Ettan MDLC
18-1176-44, 11-0008-41
Other products required
Finnigan LTQ mass spectrometer (Thermo Electron)
TurboSEQUEST™ protein identification software
(Thermo Electron)
BioBasic™ SCX, 2.1 x 250 mm (Thermo Electron)
Zorbax™ 300-SB C18 trap column,
300 µm i.d. x 5 mm, 3 µm (Agilent)
Zorbax 300-SB C18 analytical column,
75 µm i.d. x 150 mm, 3 µm (Agilent)
Ammonium bicarbonate (Merck)
Citric acid (Fluka)
Formic acid, ultrapure (Merck Suprapur™)
Iodoacetic acid (Merck)
Acetonitrile, HPLC grade
Water, HPLC grade
Fraction collection
on microplate
Capture of
peptides
Separation of
peptides released
from RPC trap column
RPC trap
RPC
RPC trap
RPC
SCX
Elution of peptides
by salt gradients
Fractions collected on microplates in
Fraction Collector Frac-950. After the
initial SCX separation the collected
fractions are reinjected by the
autosampler onto the high-throughput
RPC trap/analytical setup
Fig 1. Schematic of the 2D-LC setup.
Methods
Sample preparation
Approximately 0.5 mg of mouse brain tissue was prepared
according to the following procedure. The proteins were
dissolved by adding 1 ml of 9 M urea with 50 mM DTT to
the tissue and allowing it to incubate for 60 min at 20 °C.
A 1-ml aliquot consisting of 8 M urea, 250 mM TrisHCl, and
125 mM iodoacetamide, pH 8.8, was added. The mixture
was allowed to incubate for 60 min at 20 °C. A 1-ml aliquot
of this mixture was buffer exchanged with 20 mM ammonium bicarbonate, pH 7.8, on a NAP-10 desalting column.
The protein sample was digested with trypsin (concentration
ratio of 50:1) for 2–24 h. The trypsin was then inactivated
by adding formic acid to the sample.
Liquid chromatography
Using the offline 2D-LC configuration of the Ettan MDLC,
40 µg of trypsin-digested mouse brain sample was injected
onto a 2.1 x 250-mm BioBasic SCX column and eluted at
100 µl/min with a linear salt gradient of 0–30% B over
40 min (A: 20 mM citric acid, pH 2.5, 25% CH3CN; B: A + 1 M
NH4Cl). Fractions were collected twice every minute (Fig 1).
The fractions were injected onto a 0.3 x 5-mm Zorbax RPC
trap column, where they were desalted. RPC separation
was performed on a Zorbax 0.075 x 150-mm analytical
column at 250 nl/min with a linear gradient of 0–60% B over
50 min (A: 0.1% formic acid; B: 84% CH3CN and 0.1% formic
acid), a step up to 100% B, and then holding at 100% B for
10 min. To increase throughput, one pair of columns was
equilibrated while the other pair was used for analysis, a
standard preprogrammed method on the Ettan MDLC.
Mass spectrometry
A Finnigan LTQ linear ion trap was used. The MS method
consisted of a cycle combining one full MS scan with three
MS/MS events (25% collision energy) followed by an MS3
event (35% collision energy) that was triggered upon detection
of -98, -49, or -32.7 Da from the precursor (neutral loss of
phosphoric acid, charge states 1+, 2+, and 3+). Dynamic
exclusion duration was set to 30 s. The MS/MS and MS3
spectra from all the runs were searched using TurboSEQUEST
protein identification software. Modifications were set to
allow for the detection of oxidized Met (+16); carboxyamidomethylated Cys (+57); phosphorylated Ser, Thr, and Tyr
(+80); and dehydrated Ser and Thr (-18).
Results and discussion
By injecting a large amount of sample and separating it
on an analytical scale SCX column, collecting the fractions,
and then injecting these onto a nanoscale LC, the peptides
of low abundance, such as phosphopeptides, could be
detected (3). Forty micrograms of a mouse brain tissue
tryptic digest was first separated by SCX using salt gradient
elution. The chromatogram is shown in Figure 2.
Thirty of the collected fractions were further analyzed by
LC-MS using the neutral loss function. Phosphopeptides were
found in one-third of the analyzed SCX fractions as shown
in Figure 2. They mainly eluted early in the salt gradient
due to their reduced charge state (4). SCX can therefore be
used as a phosphopeptide enrichment strategy.
mAU
%B
30.0
80.0
25.0
20.0
60.0
15.0
10.0
40.0
5.0
20.0
0.0
-5.0
0
-10.0
10.0
15.0
20.0
25.0
min
Fig 2. UV trace at 215 nm from the SCX separation. The bars indicate fractions
where phosphopeptides were identified, and their relative abundance. The
green line indicates increasing percentage of mobile phase B.
Database searches were then performed on all MS/MS
spectra, and the results were used to confirm the MS/MS
searches and to find tyrosine phosphorylations. Phosphotyrosine does not lose phosphoric acid during collision in
the ion trap; therefore, sequence data from MS/MS was
used to find these phosphorylations (+80). Some possible
tyrosine phosphorylations were identified but need further
evaluation. Tyrosine-phosphorylated proteins exist in very
low abundance in cells and probably fall below the detection
limit of the method.
In total, 60 phosphorylated peptides were found originating
from 50 proteins. Some of the identified phosphorylation
sites are shown in Table 1. The proteins presented in the
table are all known phosphoproteins involved in cell growth
and cell differentiation.
685.99
100
90
80
70
Relative abundance
The phosphopeptides were found and the phosphorylation
sites identified by TurboSEQUEST database searches on
all MS3 spectra. The results were confirmed manually by
studying the raw spectra. It was important to confirm that
the charge state of the peptide was correct, that the neutral
loss ion dominated the MS/MS spectrum (Fig 4), and that
the sequence data was of high quality.
MS/MS and MS3 spectra of one previously known phosphopeptide originating from Stathmin 1 are shown in Figure 4.
A previously unreported phosphorylation site originating
from scaffold attachment factor B2 is shown in Figure 5.
Scaffold attachment factor B2 has been reported to suppress
estrogen receptors and might have an important role in
breast cancer (4).
MS/MS, neutral loss of 49
60
50
40
30
20
1012.44
359.22
10
719.51
786.39
677.05
439.16
272.19
585.52
1110.39
1197.55
899.39
1327.87 1401.65
0
200
Relative abundance
An ion chromatogram and the MS3 events (i.e. where a
neutral loss was detected) from one of the fractions are
shown in Figure 3.
400
600
800
1000
m/z
1200
1400
y1-3
359.3
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
b1-10
1012.5
MS3 on m/z 686
y1-4
472.3
y1-5
b1-6 585.5
544.3
b1-7
673.4
y1-2
272.2
b1-4
326.2
0
100
200
300
y1-7
827.6
b1-8
786.5
y1-6
698.6
b1-5
397.2
400
500
600
700
b1-9
899.7
800
b1-11
1099.4
y1-9
1045.7 y1-10
1173.5
y1-8
974.8
900
1000
1100
b1-12
1196.3
1200
1400
m/z
Fig 4. MS/MS and MS3 spectra of a phosphopeptide, ApSGQAFELILSPR,
from the phosphoprotein Stathmin 1.
100
90
70
Relative abundance
Relative abundance
90
60
50
40
30
20
80
70
MS/MS, neutral loss of 49
60
50
40
30
800.38
574.27
688.61 737.34
967.32
918.38
481.25 525.40 594.20
869.39
20
10
10
0
0
0
5
10
15
20
25
30
35
Time (min)
40
45
50
55
60
45
40
Relative abundance
35
30
25
20
15
10
5
0
0
5
10
15
20
25
30
35
Time (min)
40
45
50
55
60
65
Fig 3. A) Base peak ion chromatogram of SCX fraction B10 from the
LC-MS analysis; B) all MS3 events during this analysis.
234.12
349.23
200
65
50
Relative abundance
697.55
100
80
400
600
800
1258.43
1160.50 1327.45
1080.29
1000
m/z
1200
1453.49
1400
b1-8
691.6
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
y1-9
982.5
3
MS on m/z 698
y1-5
574.3
b1-9
y1-7
820.4
800.4
y1-2
234.2
b1-1
72.0
0
100
b1-2
169.1
200
b1-7
594.3
b1-5
412.3
b1-3 y1-3
270.2 349.2
300
400
y1-4
477.9
b1-6
525.3
500
y1-8
869.5
y1-6
703.3
600
y1-10
1053.5
700
m/z
b1-10
917.8
800
900
b1-11
1045.6
1000
b1-12
1160.5
y1-11
1124.5
1100
y1-12
1225.6 b1-13
1247.6
1200
1400
Fig 5. MS/MS and MS3 spectra of a phosphopeptide, APTAALpSPEPQDSK,
from scaffold attachment factor B2.
Table 1. TurboSEQUEST result list of the putative phosphoproteins and phosphorylation sites from the MS3 spectra of mouse brain tissue.
Protein
Sequences
Mass of
MH+ (Da)
Xcorr*
SCX fraction
Microtubule-associated protein 1B
pTPEEGGYSYEISEK
KEpSKEETPEVTK
ADpSRESLKPATK
CYTTEKK(p)SP(p)SEAR
1570
1386
1284
1539
4.3
3.4
3.2
2.8
B6
C2, C3
C2
C3
Similar to microtubule-associated
protein 2
VDHGAEI(p)TQ(p)SP(p)SR
VAIIRpTPPKSPATPK
DKVTDGISKpSPEK
1492
1558
1386
4.3
3.6
3.4
B10
C1, C2
C2
Myristoylated alanine-rich protein
kinase C substrate
LSGFpSFK
LSGFpSFKK
VNGDApSPAAAEPGAK
DLpSLEEIQK
ApSGQAFELILSPR
SKE(p)SVPDFPLpSPPK
RApSGQAFELILSPR
767
895.5
1337
1057
1371
1510
1527
1.8
2.9
3.0
2.7
3.9
4.2
3.9
B7
B11
B7
B7
B7
B10, B11
B11
MARCKS-like protein
LSGLpSFK
LSGLpSFKR
AAApTPESQEPQAK
GDVTAEEAAGApSPAK
GEVAPKEpTPK
GEVAPKEpTPKK
733
896
1309
1355
1038
1166
1.6
2.9
3.0
4.3
3.4
2.3
B7
B11
B7
B7
B10
C2
Scaffold attachment factor B2
APATAALpSPEPQDSK
1394
3.9
B7
Erythrocyte protein band 4.1,
isoform 1
RSEAEEGEVR(p)TP(p)TK
1571
3.9
C4
Stathmin 1, leukemia-associated
phosphoprotein
* Cross-correlation score
Conclusions
The strategy for analyzing phosphopeptides confidently is
summarized here:
1. 2D-LC (SCX/RPC)
2. MS3 on all peptides that show neutral loss of
phosphoric acid
3. TurboSEQUEST searches on all MS3 spectra (-18@ST)
4. Manual confirmation of charge state and that neutral
loss dominates MS/MS spectra
5. Further confirmation by MS/MS searches (+80@STY)
To confidently assign phosphopeptides in a complex mixture
such as a tryptic digest of brain tissue, two-dimensional
separations are needed. 2D-LC separated the peptides with
high resolution, and the neutral loss MS detection was very
selective for phosphopeptides. Care had to be taken when
interpreting the data to avoid false positives from the
database searches.
To increase the number of identified phosphopeptides, a
greater amount of starting material would be needed (5),
and possibly another chromatographic enrichment step
specific for phosphopeptides, for example using titanium
oxide media (6).
References
1.
Ficarro, S. B. et al. Phosphoproteome analysis by mass spectrometry and its
application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).
2.
Application note: Highly sensitive phosphopeptide analysis using Ettan MDLC
and a linear ion trap mass spectrometer, GE Healthcare, 11-0027-38, Edition
AA (2005).
3.
Beausoleil, S. A. et al. Large-scale characterization of HeLa cell nuclear
phosphoproteins. Proc. Natl. Acad. Sci. USA 101, 12130–12135 (2004).
4.
Jiang, S. et al. Scaffold attachment factor SAFB1 suppresses ERα-mediated
transcription in part via interaction with N-CoR. Mol. Endocrinology
10.1210/me.2005-0100 (29 September 2005).
5.
Ballif, B. A. et al. Phosphoproteomic analysis of the developing mouse brain.
Mol. Cell. Proteomics 3, 1093–1101 (2004).
6.
Pinkse, M. W. et al. Selective isolation at the femtomole level of phosphopeptides
from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide
precolumns. Anal. Chem. 76, 3935–3943 (2004).
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