AMINO ACID ANALYSIS OF

PHOSPHO-PEPTIDES: ABRF-93AAA
K. Ümit Yüksel l, Thomas T. Andersen 2, Izydor Apostol3, Jay W. Fox 4,
John W. Crabb 5, Raymond J. Paxton 6 and Daniel J. Strydom 7

1 Dept. Biochem. & Mol. Biol., U. North Texas Health Sci. Ctr. Ft Worth, Ft. Worth, TX 76107
2 Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, NY 12208
3 Somatogen Inc., Boulder, CO 80301
4 Department of Microbiology, University of Virginia Medical School, Charlottesville, VA 22908
5 Protein Chemistry Facility, W. Alton Jones Cell Science Center, Lake Placid, NY 12946
6 Department of Protein Chemistry, Immunex Corp., Seattle, WA 98101
7 Center for Biochem. Biophys. Sciences and Medicine, Harvard Medical School, Boston, MA 02115

I. INTRODUCTION
The Association of Biomolecular Resource Facilities (ABRF) exists to serve the
analytical and synthetic needs of laboratories which study and use proteins, nucleic
acids and other macromolecules. Part of ABRF’s mission is educational, assisting
member laboratories to achieve and maintain the highest quality of services
possible. The ABRF Amino Acid Analysis Research Committee has carried out
annual studies since 1989 [1-6] as a means for members to readily assess their
performance relative to other core facilities. The aim of this year’s study was to
evaluate amino acid analysis methods for identification and quantitation of post-
translationally modified residues with emphasis on phosphorylated amino acids
(Ser, Thr, and Tyr) and on hydroxyproline.
II. MATERIALS AND METHODS
A. Sample Preparation and Distribution
The 1993 ABRF amino acid analysis test sample (ABRF-93AAA) was a 30
residue synthetic peptide with the following sequence and four intended
phosphorylation sites:
L(pS)G(pY)A(pT)F(pS)(hP)LDTQSLLKIYNTPLSVIISDK.
This peptide was designed to provide both phosphorylated and unmodified Ser, Thr
and Tyr. In addition, a particularly slow cleaving bond (Ile-Ile) was included to
provide a hydrolysis challenge. The peptide was synthesized by the University of
Michigan Protein and Carbohydrate Facility on a preloaded Wang resin using Fmoc
chemistry. The residues to be phosphorylated were introduced with free side-
chains. The resin bound peptide was base treated and then globally phosphorylated
(7), using a five-fold molar excess of di-t-butyl phosphoramidate in dry N-
methylpyrrolidone (NMP). Oxidation was with t-butylhydroperoxide in NMP.
TECHNIQUES IN PROTEIN C
Copyright © 1994 by Academic
All rights of reproduction in any form reserved.
232 K. Ümit Yüksel

The peptide was deprotected and cleaved (TFA:EDT:thioanisol =90:5:5), and

purified by RP-HPLC (Polymer Labs PLRPS) using an acetonitrile gradient in
0.1% NH4OH. The sequence of the peptide was partially confirmed by automated
Edman degradation. No calls could be made at positions 28 (Ser), 29 (Asp) and 30
(Lys, C-terminus). Electrospray mass spectrometric analysis (ESMS) of the
sample provided a mass of 3542 Da for the major peptide, indicating incomplete
phosphorylation at the 4 sites. The expected masses for the peptide with 3 and 4
phosphates are 3541.7 and 3621.7 Da, respectively. ESMS analysis of lysyl and
aspartyl proteolytic cleavage products from ABRF-93AAA showed that the
predominant species of both N-terminal phosphopeptides contained 3 phosphates,
however a significant amount of tetra-phosphorylated peptides were also present.
The precise level of phosphorylation of ABRF-93AA was not determined.
Extensive amino acid analysis was also conducted by the authors. The preparation
was dissolved in 0.1% N-ethylmorpholine/50% acetonitxile, the concentration
determined by amino acid analysis, and ~23 µ g (6.47 nmol) aliquots of dried
peptide distributed to 233 ABRF facility directors for amino acid analysis using
methods of their choice. Participants were told that the sample contained ~20 µg
(5-6 nmol) of a synthetic peptide and they were asked to report the yield (pmol) of
all amino acids found, including phosphorylated residues and unusual amino acids.
In addition, answers were requested to specific questions regarding methodology,
calibration and quantification. A brief outline of phosphoamino acid analysis
methods and references were provided with the sample.
B. Calculations
Raw data were received by an independent collaborator, identifying marks
removed and the anonymous results forwarded to the 1993 ABRF Amino Acid
Analysis Research Committee. Data reduction was performed as previously
described [2]. The amount of peptide (pmol) was calculated from its known
content of Asp, Glu, Ala, Gly, Pro, Thr, Ser, Tyr, Val, Ile, Leu, Lys and Phe,
excluding values differing more than 15% from the average [2]. The accuracy of
each residue was calculated as % Error, and the overall accuracy of the composition
as the Average % Error, where
% Error = 100 x (experimental value - true value) / (true value)
Average % Error = ( ∑ |% Error for 13 amino acids|) / 13.
Errors associated with phosphorylated residues and hydroxyproline were not
included in the calculation of average percent error.
III. RESULTS AND DISCUSSION
Fifty sites participated in this study, which represents a 23% participation level
from the 214 ABRF member laboratories listing amino acid analysis as a service or
research activity. Due to an obvious gross contamination by extraneous material,
one of the 50 sites was not considered further. The remaining 49 sites
encompassed 36 which attempted the analysis of phosphoamino acids, and 15 sites
correctly identified the presence of Hyp.
Analysis of Phospho-Peptides 233

TABLE I. Methods and instrumentation used to analyze ABRF-93 AAA

Instrumentation
Common Amino Acids common Amino Acids Phosphoamino Acids
-column 32 Pre-column 2 Pre-column 25
PTC 29 ABI 420 2 . . . . . . . . . . . . . . . . . . . . . . . 2.
OPA/FMOC 2 ABI 420A 10 ........................ 7
AQC* 1 ABI 420H 2 ........................ 3
ost-columQ 18 Beckman 1l0B 1 Beckman126 1
ninhydrin 15 Beckman 421A 1 BeckmanPACE † 1
OPA 3 HP 1090 L 1 ........................ 1
HP 1090 M 1
Phosphoamino Acids HP 1090 AQII 2 ........................ 2
-column 25 Waters Pico-tag 9 ........................ 5
PTC 19 ........................ 1
OPA/FMOC 2 Waters 625 1 ........................ 1
AQC* 1 Waters 1 ........................ 1
DABSYL 3 Post-column 17 Post column 11
Beckman 6300 14 ........................ 7
Post-column 11 ISCO 2360 1 ........................ 3
ninhydrin 8 JEOL 300 1 ........................ 2
OPA 3 Waters 1 ........................ 1
* AQC: 6-aminquinolyl-N-hydroxysuccinimidyl carbamate
† Capillary zone electrophoresis

A. Instrumentation
The participants analyzed ABRF-93AAA mostly by pre-column derivatization
procedures (32/49 = 65%; Table I). This value is higher than the last five studies
[2-6], and suggests a trend towards increased use of pre-column methods (49% of
the ABRF-89AAA participants used pre-column methods). Derivatization with
PITC and ninhydrin were the preferred pre- and post-column methods. Three sites
analyzed phosphoamino acids by the pre-column DABSYL technique, but used the
ninhydrin method to detect the common amino acids. Analyses were performed
with the instrumentation summarized in Table I.
B. Calibration
The average age of the common amino acid standards used to calibrate the
chromatograms was 67 days (median 30 days). In contrast the average age of the
phosphoamino acids was only 9.5 days (median of 4 days). Several facilities used
more than one type of standard for calibration. Forty-one sites calibrated their
analyses on free amino acids, 13 sites (also) calibrated on hydrolyzed free amino
acids, and 8 sites on hydrolyzed peptides. In general, the use of hydrolyzed
standards did not correlate with the quality of analyses, although the best two sites
used hydrolyzed proteins in calibration.
234 K. Ümit Yüksel

173
25
m Pre-column PTC
m Pre-column OPA/FMOC
m Pre-column AQC
0 Post-column ninhydrin
m Post-coiumn OPA

0
45 50 25 41 10 35 9 18 23 46 27 48 47 13 24 7 31 8 29 21 4 39 12 11 34
3 8 4 9 3 2 2 6 6 2 33 5 16304236221720 3 19 1 1 5 1 4 3 7 4 0 4 3 4 4
Site
Figure 1. The absolute yields of ABRF-93AAA. The average yield of sample (in
nmol) was calculated from the amino acid composition as described in the text.

Twenty-nine sites calibrated their analyses on free phosphoamino acids, while 9

sites (also) calibrated on hydrolyzed free phosphoamino acids, and 3 sites
hydrolyzed a known phosphopeptide for calibration. Calibration on hydrolyzed
phosphoamino acids yielded higher values for these compounds in most cases
(0.63 vs. 0.39 mole pSer/mole peptide). Hydrolysis of free amino acids produces
different rates of degradation compared to hydrolysis of intact proteins/ peptides
[8]. Therefore, calibration from a hydrolyzed phosphopeptide standard (if
available) probably is the better approach to improved quantitation.
C. Overall Yield and Accuracy
The average peptide yields obtained by the participating facilities are shown in
Fig. 1
23.8 nmol. When 10 sites are eliminated from both extremes, this value narrows
considerably to 7.0 +/- 0.7 nmol. The average peptide yields from these central 29
sites were very similar, and within 8% of the amount distributed (6.47 nmol).
The average % error obtained by each participating laboratory in the analysis of
common amino acids in ABRF-93AAA is plotted in Fig. 2 and summarized by
methodology in Table II. Overall, the post-column techniques were more accurate
and had a narrower error range. Eighty-two percent (14/17) of the sites with errors
greater than the overall average error were pre-column sites. Despite these
differences, the lowest error was similar for both techniques and obtained using a
pre-column method (pre-column 3.1 % and post-column 4.4 % error).
Analysis of Phospho-Peptides

30
lPre-column P T C
m Pre-column OPA/FMOC
5 20
5 l Pre-column AQCC
l Post-column ninhydrin

E
l Post-column OPA

8
$ 10
n

17 29 23 30 6 25 15 7 1 36 44 4 49 3 42 2 9 20 43 10 33 18 21 31 40
46 27 13 8 16 39 41 48 26 19 24 14 34 5 35 47 22 12 38 32 11 45 50 37

Site
Figure 2. The quality of the analyses of ABRF-93AAA. Average % errors of
amino acid composition determined by each site excluding the phosphoamino acids.

TABLE II. Correlation of error for common amino acids with technique and
sample load

% error
average range n average range n
overall 12.5 +/- 6.7 3.1-29.9 49 1.68 +/-2.05 0.05-11.41 49
Post-column † 9.7+/- 4.5 4.4-20.7 18 2.72 +/-2.81 0.23-11.41 18
ninhydrin 10.0+/- 4.9 4.4-20.7 15 2.86 +/-3.04 0.23-11.41 15
OPA 8.4+/-1.8 6.7-10.3 3 2.00 +/-0.79 1.14 - 3.05 3
Pre-column † 14.1 +/-7.2 3.1-29.9 31 1.07 +/- 1.04 0.05-3.81 31
PTC 13.7 i6.9 3.1-28.9 28 1.16 +/- 1.02 0.05-3.81 28
OPA/FMOC 22.0 14.0-29.9 2 0.22 0.09-0.34 2
AQC 8.8 1 0.23 1

†The difference between these values is significant, p <0.05, Student’s t-test.

The difference in the observed error could be due to the amount of sample analyzed.
PTC sites generally used a smaller amount (Table II). The correlation between
comparatively larger error for the precolumn assays with the smaller amounts
hydrolyzed were also noted in a previous ABRF study [2]. The average error for
PTC sites decreased from 16% to 11% when the amount of sample hydrolyzed was
increased from -0.5 to 5.0 µg. This study [2] also demonstrated that when similar
amounts of protein were hydrolyzed, the average errors were similar for both ion
exchange/ninhydrin and PTC amino acid analysis (i.e. ~16-47% and 9-11%,
respectively). The magnitude of % error by residue is reviewed in Fig. 3. The high
error in PTC-Glu may be related to its co-elution with PTC-Hyp (see below).
236

60 l

l Pre-column
l post-column

a A l a A s p Glu Gly Ile L e u L y s P h e P r o Ser Thr Tyr Val Hyp

Amino Acids
Figure 3. Effect of methodology on accuracy. The average residue error’ (%) was
calculated as ( ∑ | error |)/number of sites. Bars represent standard deviation from the mean.

D. Analysis of Phosphoamino Acids

The majority of sites hydrolyzed the sample for short periods of time (1-4 h)
and analyzed it with either their standard analytical system, or with some
modification of the chromatography. Phosphoserine was identified by 28 sites,
pThr by 25, and pTyr by 29. In addition, some sites could not separate the
phosphoamino acids from each other, and reported them in combination.
Conversion of pSer to S-ethylcysteine was performed by two sites yielding 0.75
mol pSer/mol peptide for one PTC-site, and nothing for another post-column OPA-
site.
The analytical data on phosphoamino acids are summarized in Table III. The
wide range of these residues that were reported, points to the difficulty in
accomplishing quantitative analyses of these amino acids (Fig. 4). The correlation
of phosphoamino acid identification with methodology is marked. The presence of
phosphorylated residues was determined by 77% of the sites attempting analysis
with pre-column methods, but only 56% of the sites attempting analysis with post-
column methods. The co-elution of phosphoamino acids made it difficult to
identify specific amino acids with the ion-exchange methodology. However, the
quantitative recovery for the phosphoamino acids appears to be independent of pre-
or post-column technology (Table III). The separation of phosphoamino acids by
pre-column PITC techniques is rather straightforward (Fig. 5), but requires minor
adjustments in the chromatographic setup. The ion exchange techniques are
cumbersome in this respect (Fig. 5). In some cases the starting buffer may have to
be adjusted to a lower pH and the temperature dropped significantly (10oC). In
other cases the complete separation may take several hours. Representative
chromatograms for pre- and post-column separation of phosphoamino acids are
Analysis of Phospho-Peptides 237

shown in Fig. 5.
Modified hydrolysis conditions are required for the analysis of phosphoamino
acids. In this study, the shortest time of hydrolysis (≤ 60 min at 110OC) generally
gave the highest amount of pSer and pTyr, while the yield of pThr peaked at later
times (≥ 120 min at 110oC). Low but quantifiable amounts of pThr remained after
most standard hydrolysis conditions.

Table III. Yields of phosphoamino acids *

Residue Theoretical † Average Range Pre-column (n) Post-column (n\

pSer 2 0.60+/-0.27 0.1-1.27 0.57+/-0.27(20) 0.66+/-0.26(8)
PThr 1 0.39+/-0.22 0.1-0.99 0.40+/-0.24(17) 0.39+/-0.18(8)
pTyr 1 0.60+/-0.53 0.03-2.32 0.66+/-0.59(21) 0.45+/-0.30(8)
overall 1.29+/-0.83 0.13-3.78 1.28+/-0.85(25) 1.33+/-0.77(9)

* Sites reporting zero yield were not included, units are moles/mole peptide.
† Although the theoretical level of phosphorylation was 4 moles /mole peptide, ESMS indicated
that the peptide contained less than 4 moles phosphate / mole peptide

E 1122333540424345 3 1 44 7 151248144147 613242921461020 8 2338 5 493717

m Post-column OPA

5152145 312 6 14440334122 747372920143023493542481324 04310111746

Site

Figure 4. The yields phosphoamino acids of ABRF-93AAA.

Only sites reporting the yield of at least one of the phosphoamino acids are shown. Sites which
used hydrolyzed standards are marked with (*).
238 K. Ümit Yüksel

Figure 5: Chromatography of phosphoamino acids.

Top: FTC-phosphoamino acids were resolved by RP-HPLC on an Inertsil ODS2 column
(4.6x150 mm, 5 pm).Separation was achieved at 1 mL/min with a gradient of solvent A (100
mM sodium acetate /2.5% acetonitrile, pH 7.75) against solvent B (60% acetonitrile):0-35%B
in 12 min. 35-90%B in 13 min., isocratic for 0.5 min, l00%B in 4.5 min. The sample
consisted of Amino Acid Standard H (Pierce) supplemented with pSer, pThr, pTyr and S-ethyl-
cysteine, 200 pmol each. The mixture was gas phase hydrolyzed in 6 N HCI for 1 h at 110oC.
Bottom:Phosphoamino acids (Sigma) were resolved by ion exchange chrmatography on a
Pierce AA511 column, isocratically developed at 57oC with Buffelute A (Pierce) diluted 1:1
with water, and adjusted to pH 1.50. Flow rate: 0.4 mL/min. Sample ws applied in 0.1 %
TFA/water (pH 1.06).
Analysis of Phospho-Peptides 239

E. The Unknown Amino Acid: Hydroxyproline

Hydroxyproline was included in the peptide to test the general capability of core
facilities to recognize and identify an unknown. Twenty sites (41%) reported the
presence of the extra unknown, despite the confounding presence of phosphoamino
acids. Hydroxyproline was correctly identified and quantified by 15/49 sites
(31%), while a few sites misidentified it and two sites reported it as an unknown.
The methodology (pre- vs. post-column derivatization) did not make a difference in
the success for recognition of this unknown (7/18 =41% post; 13/31 = 41% pre).
Based on the markedly high values of pTyr and the absence of pSer and pThr, four
additional PTC-sites may have misidentified Hyp as pTyr. This is not surprising
since these two amino acids can elute very closely in some systems. Another
complication is the co-elution of Hyp and Glu in some standard PTC separation
systems. Thus, 6 PTC-sites and 1 OPA/FMOC site did not detect Hyp while their
Glu content was markedly high, most likely due to Hyp and Glu co-eluting.
Notably, pSer, pThr, pTyr and Hyp and the common 16 amino acids in protein
hydrolyzates can be well resolved in most PTC analysis systems by adjusting the
pH of the chromatography solvents (e.g. from pH 5.9 to 6.4 for the ABI system).
IV. SUMMARY AND CONCLUSIONS

In response to the increasing need for identification and quantitation of post-

translationally modified amino acids, ABRF has provided its members with
peptides containing phosphorylated and hydroxylated amino acids [9]. ABRF-
93AAA is a sample containing hydroxyproline and phosphorylated and unmodified
Ser, Thr and Tyr. Fifty facilities participated in this study which produced 49 sets
of useable data. These investigators have utilized diverse chemistries (AQC,
DABSYL, ninhydrin, OPA, OPA/FMOC, PITC) and chromatographic conditions
(ion exchange, reversed phase, and capillary electrophoresis). The best results
(three sites with <5% error) are shown in Table IV. The study has provided a
useful comparison of phosphoamino acid identification methods, although the
precise amount of phosphate in the sample was not determined, Resolution of
phosphorylated amino acids is more readily achieved by pre-column techniques,
and these sites were more successful in their identification of phosphoamino acids
than post-column sites (77% vs. 56%). The fact that 60% of the participants were
able to identify the phosphoamino acids in the sample emphasizes the feasibility and
screening value of phosphoamino acid analysis. The results indicate that
identification of common amino acids and phosporylated amino acids must be made
on separate hydrolyzates. Short hydrolysis times (e.g. 2 h at1l0oC) provide a
better yield of the phosphorylated amino acids. Longer hydrolyses(2 24 h) are
required to obtain accurate values for common amino acids, especially for slow
cleaving bonds like Ile-Ile.
Hydroxyproline was equally well identified by pre- and post-column
techniques. Hydroxyproline was less frequently identified than the phosphoamino
acids, in part due to its co-elution with phosphotyrosine and/or Glu in some PTC-
sites, as well as its unannounced presence.
240 K. Ümit Yüksel

Table IV. Best analysis of common amino acids of ABRF-93AAA.

Site / Chemistry I
17 / PITC 46 / PITC 29 /ninhydrin Overall
. e o r . † Pre-Column Pre-Column PostColumn Avrg. +/- St.Dev.
Ala 1 1.02 1.03 1.01 1.10 +/-0.13
Arg 0.03 0.04 0.00 0.11+/-0.19
Asp 3.02 3.08 3.34 3.02+/-0.60
Glu 1.02 0.88 0.98 1.13+/-0.30
Gly 1.04 1.00 1.02 1.14+/-0.26
His 0.03 0.06 0.00 0.06+/-0.18
Ile 2.95 3.15 2.84 2.68+/-0.32
Leu 4.83 5.44 5.14 4.92+/-0.47
Lys 1.96 1.87 2.00 2.03+/-0.35
Met 0.03 0.01 0.00 0.05+/-0.09
Phe 1.00 0.97 1.01 1.02+/-0.13
Pro 1.01 1.03 1.00 1.07+/-0.12
Ser* 4.04 5.02 4.70 4.18+/-0.63
1.98 2.08 2.48 2.20+/*-0.28
Tyr* 1.96 1.97 1.95 1.78+/-0.20
Val 1.01 0.96 0.99 0.97+/-0.28
Hyp 0.98 0.86+/-0.20
Pser 0.96 0.58 0.53 0.60+/-0.27
Pthr 0.99 0.39 0.35 0.39+/-0.22
Ptyr 1.83 2.32 0.37 0.60+/-0.53
Amt. hydrolyzed (µg) 2.6 1.93 2.77
Amt. analyzed (µg) 0.65 0.77 1.39
Total Yield (µg) 26 23.2 27.7
Error(%) 3.05 4.09 4.43
† Residues per mole of peptide.
* Values expected after acid hydrolysis upon relase of phosphate group are in paranthesis.

Acknowledgements
We would like to thank to Dr. Yongde Bao (U. Virginia) for receiving the data and maintaining the
anonymity of the collaborating facilities. We also thank Dr. Phil Andrews (U. Michigan), and K.
West & C. Johnson (W. Alton Jones Cell Science Center) for expert assistance in the preparation
and distribution of the sample, and to all the ABRF facilities who have taken part in this project.
This work was supported in part by NSF grant DIR 9003100 (to JWC) on behalf of the ABRF
References
1. Niece, R.L., Williams, K.R., Wadsworth, C.L., Elliot, J., Stone, K.L., McMurray,W.J.,
Fowler, A., Atherton, D., Kutny, R., and Smith, A. (1989) in “Techniques in Protein
Chemistry” (T.E. Hugli, ed.), Academic Press, San Diego, pp 89-101.
2. Crabb, J.W., Ericsson, L.H., Atherton, D., Smith, A.J. and Kutny, R. (1990) in “Current
Research in Protein Chemistry” (JJ. Villafranca, ed.) Academic Press, San Diego, pp 49-61.
3. Ericsson, L.H., Atherton, D., Kutny, R., Smith, A.J. and Crabb, J.W. (1991) in ‘Methods of
Protein Sequence Analysis 1990” (H. Jörnvall and J.-O. Höög eds.) Birkhauser Verlag, Basel.
4. Tarr, G.E., Paxton, R.J., Pan, Y.-C.E., Ericsson, L.H., and Crabb, J.W. (1991) in Techniques
in Protein Chemistry II” Academic Press, pp 139-150.
5. Strydom, D.J., Tarr, G.E., Pan, Y.C.E., and Paxton, R.J. (1992) in “Techniques in Protein
Chemistry III” (R.H. Angeletti, ed.) Academic Press, San Diego, pp 261-274.
6. Strydom, DJ., Andersen, T.T., Apostol, I., Fox, J.W., Paxton, R.J., and Crabb, J.W. (1993) in
‘Techniques in Protein Chemistry IV” (R.H. Angeletti, ed.) Academic Press, San Diego, pp 279-
288.
7. Andrews, D.M., Kitchen, J., and Seale, P.N. (1991) lntl. .J. Peptide Protein Res. 38,469-475.
8. Bylund, D.B., and Huang, T.S. (1976) Anal. Biochern. 73,477.485.
9. Mische, S.M., Yüksel K.U. Mende-Mueller, L.M., Matsudaira, P., Crimmins, D.L., and
Andrews, P.C. in “Techniques in Protein Chemistry IV”, R. Angeletti, ed., Academic Press, San
Diego, 1993,453-461.