Beruflich Dokumente
Kultur Dokumente
Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055
tography/MS and matrix-assisted laser desorption/ionizationMS.20 This approach is based on specific chemisorption of
phosphate groups on the surface of titanium oxide and was shown
to result in less nonspecific binding than IMAC. In addition,
column preparation is more straightforward, eliminating the metal
ion chelating and washing steps required for preparation of IMAC
columns. An alternative strategy involving magnetic Fe3O4/TiO2
core/shell nanoparticles as affinity probes for phosphopeptides
has also been recently demonstrated.21 Finally, utilization of a
metal hydroxide, Al(OH)3, for phosphopeptide and phosphoprotein
binding also proved effective and more selective than commercial
phosphoprotein enrichment kits.22 Here, we present enrichment
of phosphorylated peptides on the surface of zirconium dioxide
loaded in a microtip. ZrO2 has drawn attention as chromatographic
column material due to its physical (e.g., toward extremes of pH)
and thermal stability and its unique surface chemistry.23-25
However, to our knowledge, its use for phosphopeptide isolation
has not previously been reported in the literature. We investigated
the performance of these microtips for trypsin and Glu-C proteolytic digests of R- and -casein and compared ZrO2 phosphopeptide binding specificity and recovery to TiO2-based enrichment.
Mass spectrometric phosphopeptide analysis was performed with
electrospray ionization (ESI) in both negative and positive ion
modes with a Fourier transform ion cyclotron resonance (FT-ICR)
instrument.
EXPERIMENTAL SECTION
Reagents and Sample Preparation. R-Casein and -casein
from bovine milk (Sigma, St. Louis, MO) were prepared in 25
mM ammonium bicarbonate (Fisher Scientific, Fair Lawn, NJ)
buffer to a concentration of 25 M. Trypsin (Promega, Madison,
WI) digestion was performed for 15 h at 37 C at an enzyme/
substrate ratio of 1:50. Glu-C (Roche, Penzberg, Germany)
digestion also proceeded for 15 h but at 25 C at an enzyme/
substrate ratio of 1:100. Microtips filled with ZrO2 and TiO2 (25
or 50 g) were either provided as a gift or purchased from Glygen
(Columbia, MD) and used without further modification. For
optimization of the phosphopeptide enrichment conditions, binding
solutions of different pH were prepared by adding various
concentrations (see below) of formic acid (Acros Organics, Fair
Lawn, NJ) or ammonium bicarbonate to HPLC grade water
(Fisher). Washing solutions consisted of pure water (pH 6.5), 250
mM ammonium acetate (pH 6.9), 100 mM ammonium bicarbonate
(pH 8.0), or 75 mM ammonium hydroxide (pH 10.5). Finally, 0.5%
piperidine (pH 11.5) or 0.25 or 0.5% ammonium hydroxide (pH
10.8 and 11, respectively) were used for eluting bound phosphopeptides. Peptide mixtures from the enzymatic digests were
fractionated into 1-100-pmol aliquots, dried down in a vacuum
concentrator (Eppendorf, Hamberg, Germany), reconstituted in
binding solution, and loaded onto microtips that had been
equilibrated with the same binding solution. Unbound peptides
(21) Chen, C. T.; Chen, Y. C. Anal. Chem. 2005, 77, 5912-5919.
(22) Wolschin, F.; Wienkoop, S.; Weckwerth, W. Proteomics 2005, 5, 43894397.
(23) Nawrocki, J.; Rigney, J.; McCormick, A.; Carr, P. W. J. Chromatogr., A 1993,
657, 229-282.
(24) Nawrocki, J.; Dunlap, C.; McCormick, A.; Carr, P. W. J. Chromatogr., A
2004, 1028, 1-30.
(25) Hoth, D. C.; Rivera, J. G.; Colon, L. A. J. Chromatogr., A 2005, 1079, 392396.
1744
1745
Table 1. List of Phosphopeptides Found in Negative Mode FT-ICR Mass Spectra from Proteolytic Digests of r- and
-Casein without (w) Phosphopeptide Enrichment and Following ZrO2 and TiO2 Enrichment
no.
peptide identity
enzyme
phosphorylation
monoisotopic
mass
method
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
R-casein-S2:153-164
R-casein-S2:141-152
R-casein-S2:152-164(153-165)
R-casein-S1:121-134
R-casein-S1:58-73
R-casein-S1:58-73
R-casein-S1:119-134
R-casein-S1:118-134
R-casein-S1:74-94
R-casein-S2:16-36
R-casein-S2:61-85
R-caseinS2:57-95
-casein:48-63
-casein:45-63
-casein:17-40
-casein:16-40
-casein:16-40
R-casein-S1:126-133
R-casein-S1:55-65
R-casein-S1:126-140
R-casein-S1:55-70
R-casein-S1:77-92
R-casein-S2:67-83
R-casein-S2:16-33
-casein:47-52
-casein:47-57
-casein:47-59
-casein:21-36
-casein:21-46
-casein:21-46
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
trypsin
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
Glu-C
1phos:158
2phos:144,146
1phos:158
1phos:130
1phos:61,63,68a
2phos:61,63,68a
1phos:130
1phos:130
1phos:79
4phos:23,24,25,31
4phos:71,71,73,76
4phos:71,72,73,76
1phos:50
1phos:50
4phos:30,32,33,34
3phos:30,32,33,34a
4phos:30,32,33,34
1phos:130
2phos:61,63
1phos:130
3phos:61,63,68
5phos:79,81,82,83,90
4phos:71,72,73,76
4phos:23,24,25,31
1phos:50
1phos:50
1phos:50
4phos:30,32,33,34
3phos:30,32,33,34a
4phos:30,32,33,34
1465.6047
1538.5902
1593.6997
1659.7868
1846.7179
1926.6842
1950.9451
2079.0401
2719.9055
2745.9923
3007.0221
4717.9274
2060.8211
2431.0430
2965.1571
3041.5000
3121.2582
937.3793
1324.4836
1818.8335
1978.6556
2075.6103
2093.6080
2353.7863
846.3160
1460.5820
1704.6516
2007.6804
3110.4225
3190.3888
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
Zr, Ti
Zr, Ti
w, Zr, Ti
w, Zr, Ti
Zr
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w
w, Zr, Ti
w
w, Zr, Ti
w
w
w, Zr, Ti
Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
w, Zr, Ti
ZrO2 enrichment (74 and 31%, respectively). In addition, the 4charge state of peptide 7 is not detected following TiO2 enrichment, nor is the singly phosphorylated peptide 4. These enrichments were performed from 100-pmol aliquots of the same tryptic
digest. Thus, variations in the digestion conditions cannot explain
the absence of peptide 4. We believe these results constitute an
important finding because a plentitude of biological regulatory
mechanisms involve a single phosphorylation event and our results
imply that ZrO2 phosphopeptide enrichment would be preferred
over other isolation strategies in the characterization of such cases.
We do not believe the observed behavior is a result of irreversible
binding of multiply phosphorylated peptides to ZrO2 because we
could still detect such phosphopeptides in the left-over solution
of tryptic peptides. Also, we do not believe the differentiation
between singly and multiply phosphorylated peptides is a result
of our specific enrichment protocol because changing the conditions did not result in varied ratios of, for example, peptides 6
and 7 for R-casein (Figure 1).
The finding that ZrO2 more selectively isolates singly phosphorylated peptides as compared to TiO2 was confirmed with a
tryptic digest of -casein; see Figure 2d-f. Again, the most
abundant peak following ZrO2 enrichment originates from a singly
phosphorylated peptide (the 2- charge state of FQSEEQQQTEDELQDK, corresponding to amino acid residues 48-63 in
-casein (peptide 13 in Table 1) whereas its abundance following
TiO2 enrichment is comparable to the quadruply phosphorylated
peptide RELEELNVPGEIVESLSSSEESITR (peptide 17, triply
deprotonated). The latter peptide displays similar S/N ratios
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Figure 2. Negative mode ESI FT-ICR mass spectra (8 scans) from 100 pmol of tryptic digests of R- and -casein obtained prior to phosphopeptide
enrichment (a, d) and following ZrO2 (b, e) and TiO2 phosphopeptide enrichment (c, f). Phosphopeptides were bound in 3.3% formic acid (pH
2), washed with water, and eluted in 0.5% piperidine (pH 11.5). Highly selective enrichment of singly phosphorylated peptides (7 and 13, see
Table 1) is observed following ZrO2 enrichment. Nonphosphorylated peptides observed following enrichment are labeled with their corresponding
amino acid residue numbers and isoform (for R-casein). *, noise.
phosphopeptides), hence reducing nonspecific binding. We explored the use of Glu-C digestion prior to ZrO2 and TiO2
enrichment; see Figure 3. For R-casein (Figure 3a-c), Glu-C
digestion resulted in lower overall (i.e., with or without enrichment) phosphopeptide signal as compared to trypsin digestion
(see Table 2). With TiO2, the enrichment factor (i.e., the increase
in relative phosphopeptide signal) following Glu-C digestion was
about the same (2.5-fold) as for the R-casein trypsin digest
whereas a higher enrichment factor (4-fold relative increase)
was observed with ZrO2 for the Glu-C digest (compared to 2.5fold relative increase for the trypsin digest). From repeated
experiments, the error of the tabulated selectivity values was
estimated to be 8%. Again, one additional singly phosphorylated
peptide (peptide 20 in Table 1) was detected following ZrO2
enrichment as compared to TiO2 enrichment. However, several
multiply phosphorylated peptides (peptides 22-24 in Table 1)
detected prior to TiO2 and ZrO2 treatment were not observed
following the use of metal oxide microtips, rendering trypsin
digestion the preferred strategy for R-casein.
For -casein, the enrichment factors following both TiO2 and
ZrO2 treatment were similar for the Glu-C and trypsin digests (see
Table 2). Additional evidence for the selective enrichment of singly
phosphorylated peptides with ZrO2 is evident from Figure 3e and
f: Following both ZrO2 and TiO2 enrichment, the most abundant
species correspond to the 2- charge state of singly phosphorylated
KFQSEEQQQTEDE (peptide 27 in Table 1). However, the relative
abundance is 64% following ZrO2 treatment and 44% following TiO2
treatment. Again, TiO2 shows higher selectivity than ZrO2 for a
multiply phosphorylated peptide (quadruply phosphorylated LNVPGEIVESLSSSEE, peptide 28 in Table 1): The 2- charge state
of this peptide is detected at a 27% relative abundance following
TiO2 enrichment versus 15% for ZrO2. Also, its triply deprotonated
form is only observed following TiO2 enrichment.
Sensitivity of ZrO2 and TiO2 Microtip Phosphopeptide
Enrichment. All spectra presented above were obtained following
enrichment of 100 pmol of a phosphoprotein digest employing
microtips containing 50 g of ZrO2 or TiO2. Table 3 shows the
selectivity of these microtips with decreasing amounts of an
R-casein tryptic digest (50 and 25 pmol, respectively). Again, the
error of these values was found to be 8%. It is clear that, for
R-casein, the phosphopeptide selectivity is not compromised when
the sample amount is decreased. However, because our mass
spectrometer had not been optimized for high sensitivity (e.g.,
we did not use nanospray or an ion funnel for improved ion
transmission), we obtained poor S/N values with lower than 25
pmol of sample.
To allow utilization of the ZrO2 microtips for sample amounts
compatible with proteomic applications, it will be necessary to
scale down the size of these columns. An initial attempt to half
the amount of metal oxide to 25 g resulted in improved S/N
ratios of phosphopeptides from a tryptic digest of 1 pmol of
R-casein (Figure 4). These experiments were performed with a
digest different from the one used for the experiments shown in
Figures 1 and 2. Thus, the relative abundance of the detected
phosphopeptides is somewhat different. However, again, singly
phosphorylated peptides (peptides 4 and 7) are more abundant
Analytical Chemistry, Vol. 78, No. 6, March 15, 2006
1747
Figure 3. Negative mode ESI FT-ICR mass spectra (8 scans) from 100 pmol of Glu-C digests of R- and -casein obtained prior to phosphopeptide
enrichment (a, d) and following ZrO2 (b, e) and TiO2 phosphopeptide enrichment (c, f). Phosphopeptides were bound in 3.3% formic acid (pH
2), washed with water, and eluted in 0.5% piperidine (pH 11.5). For R-casein, poorer phosphopeptide signal is observed as compared to trypsin
digestion (Figure 2) whereas similar performance is seen for -casein with selective enrichment of a singly phosphorylated peptide (27, see
Table 1) following ZrO2 treatment. Nonphosphorylated peptides observed following enrichment are labeled with their corresponding amino acid
residue numbers. *, noise.
Table 2. Selectivitya (%) of ZrO2 and TiO2 Microtips for
Phosphopeptide Enrichment of Proteolytic Digests of
r- and -Casein
without enrichment
ZrO2 enrichment
TiO2 enrichment
a
trypsin
digest of
R-casein
trypsin
digest of
-casein
Glu-C
digest of
R-casein
Glu-C
digest of
-casein
27
67
62
22
61
65
8
31
22
22
62
77
without enrichment
ZrO2 enrichment
TiO2 enrichment
aDefined
100 pmol
50 pmol
25 pmol
27
67
62
29
85
77
29
83
74
CONCLUSIONS
In our hands, both zirconium oxide and titanium oxide
microtips provide highly selective phosphopeptide enrichment
from proteolytic peptide mixtures. However, the zirconium oxide
columns possess a unique selectivity for singly phosphorylated
peptides whereas titanium oxide selectively enriches multiply
phosphorylated peptides. Thus, these two column materials have
complementary properties and the choice of phosphopeptide
enrichment strategy can be tailored according to the specific
application. For R- and -casein investigated here, the use of Glu-C
rather than trypsin for proteolytic digestion did not appear to
provide an advantage. Current commercially available 50-g
microtips are limited to sample amounts above 1 pmol although
further miniaturization of these columns appears promising.
ACKNOWLEDGMENT
This work was supported by the Searle Scholars Program and
the University of Michigan. We also thank Ashok Shukla for
valuable discussions and for providing the 25-g microtips.
1749