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2nd Amino Acid Workshop
Plasma Protein Synthesis Measurements Using a Proteomics Strategy1
H. M. H.
VAN
Eijk and N. E. P. Deutz2
Department of Surgery, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht
University, 6200 MD Maastricht, The Netherlands
ABSTRACT The analysis of the synthesis of proteins has been the subject of many studies in animals and humans.
Plasma proteins can be used as an easy accessible source of specific proteins. In this paper, an innovative method to
study the synthetic rate of plasma proteins is described. This methodology, based on the proteomics approach, enables
the direct observation of the effects of posttranslational modifications of protein synthesis and/or degradation. The
methodology is based on 1D or 2D electrophoresis and subsequent electrospray ionization liquid chromatography
mass spectrometry (ESI-LC-MS). Protein synthesis is measured in isotopically labeled peptides of the identified
proteins. This innovative method can be used to assess amino acid adequacy and safety by studying protein synthesis
and posttranslational modification of plasma proteins in more detail. J. Nutr. 133: 2084S–2089S, 2003.
KEY WORDS: plasma proteins proteomics mass spectrometry stable isotopes
The analysis of the synthesis of proteins has been the subject
of many studies in animals and humans in the fields of nutrition
and metabolism. These studies are based on the sometimes
laborious separation of proteins from plasma and tissue and the
subsequent analysis of the enrichment of specific amino acids
within these proteins, obtained during infusion of radioactive or
stable amino acid isotopes (1,2). Plasma proteins are frequently
used for this purpose because of their easy accessibility.
Determination of the biological value of a food in relation
to its amino acid composition mostly has focused on the
measurement of the flux of precursor amino acids like
phenylalanine and leucine in plasma (3,4). The disadvantage
of this method is that it only gives an overall impression of total
protein synthesis and/or degradation, although it remains
unclear which and at what rate individual proteins are synthesized. A more logical approach therefore is to study protein synthesis of specific proteins. Up until now, however, this
required the setup of a complex methodology and probably this
is the reason why plasma protein (5) and tissue protein (6)
synthesis of specific proteins has rarely been studied.
Recently, new innovative techniques have been introduced
that are based on what is now called: ‘‘proteomics’’ (7,8). Using
modern mass spectrometric techniques, more in-depth analysis
of proteins, their composition, structure and behavior has
become possible (9).
In this paper we describe a method that combines the
traditional isotope enrichment approach with the innovative
proteomics approach to the study of plasma protein synthesis in
humans. The principle of this approach (10) is to infuse stable
isotopes of amino acids in a primed-constant and continuous
infusion protocol followed by the collection of plasma and/or
tissue samples. In addition to conventional measurements of
the isotopic dilution of precursor amino acids in the samples,
mass spectrometry (MS)3 was used to analyze crudely purified
target proteins to confirm their identity and to estimate their
enrichment by measuring a protein specific peptide fraction.
This new approach incorporates a number of benefits. It is
generally applicable for all (plasma) proteins of interest because
it does not require setting up a new isolation strategy for each
new protein to be studied and requires only a very small amount
of sample. It will also give insight into the influence of the
processes of disease on protein life span and functionality
through the study of posttranslational modifications. The
problem of how to address the precursor pool can be solved
using the mass isotopomer distribution analysis (MIDA)
technique (10). Although the method is applicable also to
tissue proteins, the main focus of this paper will be to outline
the principle of studying the synthesis of plasma proteins. In
addition, the advantages of this new approach are discussed in
relation to amino acid adequacy and safety.
Description of the techniques used in proteomics
1D and 2D gel electrophoresis. Proteins can be separated
on the basis of their molecular mass and according to their pKa
1
Presented at the conference ‘‘The Second Workshop on the Assessment of
Adequate Intake of Dietary Amino Acids’’ held October 31-November 1, 2002, in
Honolulu, Hawaii. The conference was sponsored by the International Council on
Amino Acid Science. The Workshop Organizing Committee included Vernon R.
Young, Yuzo Hayashi, Luc Cynober and Motoni Kadowaki. Conference proceedings were published in a supplement to The Journal of Nutrition. Guest editors for
the supplement publication were Dennis M. Bier, Luc Cynober, Yuzo Hayashi and
Motoni Kadowaki.
2
To whom correspondence should be addressed. E-mail: nep.deutz@ah.
unimaas.nl.
3
Abbreviations used: 1D electrophoresis, separation based on molecular
weight of proteins; 2D electrophoresis, separation based first on pKa and next on
molecular weight of proteins; APCI, atmospheric pressure chemical ionization;
CRP, C-reactive protein; ESI-LC-MS, electrospray ionization liquid chromatography mass spectrometry; HPLC, high performance liquid chromatography; MALDI,
matrix assisted laser dissociation ionization; MIDA, mass isotopomer distribution
analysis; MS, mass spectrometry; QUAD, quadrupole; TOF, time-of-flight.
0022-3166/03 $3.00 Ó 2003 American Society for Nutritional Sciences.
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PLASMA PROTEIN SYNTHESIS USING PROTEOMICS
value. These features have been exploited individually in a 1D
gel electrophoresis or combined in an approach called 2D gel
electrophoresis (11,12). In the latter approach, a sample containing a mixture of proteins is applied to a gel to which ampholytes are added in a way that divides the surface of the gel
into a defined pH-range. An electric current focuses the
different proteins to a band on the gel according to their specific
pKa value. Next, this gel strip is placed on a larger gel and
placed into a tank. A buffer is added and again an electric
current is applied. The bands of the proteins separated in the
first dimension follow the buffer front moving slowly across the
gel, and separation in the second dimension takes place
through differences in mobility resulting from differences in
molecular mass.
Mass spectrometry. Development and components of the
LC-MS. The development of modern liquid chromatographic
mass spectrometers (LC-MS) dates from the beginning of the
1990s (13). Up until then, all the processes enabling a mass
analysis took place in a high vacuum. The development of
matrix assisted laser dissociation ionization (MALDI), atmospheric pressure chemical ionization (APCI) and electro spray
ionization (ESI) enabled the design of a MS system in which the
ionization process was taken out of the high vacuum. This
revolution allowed for the first time the ionization of intact
large (bio)molecules and their subsequent introduction into the
MS. At the same time, the unraveling of the human genome
resulted in the construction of databases containing most of the
protein sequences. Together with the development of the
tryptic digestion techniques (14), a new tool to study proteins
became available under the name of proteomics. In addition,
ESI especially enabled the combination of the separation power
of an LC system, known for its ability to separate polar
biological compounds, with the third dimension offered by an
MS system on a new type of instrument called LC-MS. These
LC-MS systems are generally composed of an ionization source
operated at atmospheric pressure (ESI or APCI), some type of
mass analysis system and a detection system.
Use of MS in proteomics. How can the ability of an MS
system to measure molecular masses be exploited to identify
a specific component? The answer comes from the elucidation
of the human genome, which created a database containing the
sequence and mass of all known proteins. To understand how
MS can use this information for identification, the two main
techniques applied in modern mass analyzers must be explained. One way to address the problem is to measure exact
masses. The digestion of proteins by a protease results in a
large number of protein specific peptides. Measuring the exact
mass of these peptide fragments and comparing these with
a database library results in hopefully one, but usually a few,
possible candidate proteins from which the fragment could
originate. The better the mass precision of the analyzer, the
more likely the correct candidate is identified. This type of mass
analyses is usually performed by time-of-flight (TOF) mass
analyzers, usually in combination with MALDI ionization
(MALDI-TOF). In the other major route, the tryptic peptide
fragments are first separated using LC and transferred into the
MS system on-line. In here, the molecular mass of the separated
tryptic peptide fragment is determined using a less precise
quadrupole mass analyzer after ESI ionization (ESI-QUAD).
After isolation of this so-called parent peptide, it is fragmented
in a collision cell and the masses of the resulting daughter ions
(B and Y) are determined in a consecutive quadrupole analyzer
in a process called tandem mass spectrometry or MS-MS. In
this way, a puzzle is created, which, once solved, reveals the
amino acid sequence of the parent peptide. Next, this peptide
sequence can be compared with the protein library to find out
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in which protein it fits. This process is repeated for each LCeluted peptide fragment with a strong enough response to
enable the MS-MS analysis. As a result, a list of possible protein
candidates is created, which ranking is based on the number
of identified peptide fragments. These instruments are of the
triple quadrupole type and are comparable with ion-trap instruments, in which the above functionality is combined in a
three-dimensional (3D) quadrupole.
Subsequent to the above systems, many additional types
and/or combinations of types (hybrid instruments) have been
constructed and are now commercially available. The choice
among these systems is dependent on the type of research
question to be answered but surely also on the available budget
because the price of these systems may easily rise above half
a million U.S. dollars.
In our approach, we prefer to study plasma protein synthesis
using the ESI-ion-trap-quad systems. They not only enable
quantification of peptide isotopomers, sequencing of the peptide and thus confirmation of the identity of the originating
protein, but also enable the detection and identification of
posttranslational modifications.
Identification of plasma proteins using ESI-MS
C-reactive protein (CRP) as an example: its role in relation
to several diseases. C-reactive protein (CRP) is a plasma
protein that in the healthy condition is present at 1–5 mg/L (1).
The concentration of CRP increases 100-fold after trauma or
during acute diseases like sepsis and as such it is called a positive
acute phase protein (15). In chronic disease, the plasma CRP
level is increased to levels between 5 and 15 mg/L. This small
increase has been related to chronic inflammation associated
with these diseases (1,16). In addition, increased plasma CRP
levels have also been related to heart disease and atherosclerosis (17), and CRP is also known to exhibit antimicrobiological
activity (18). Considering all these different involvements in
acute and chronic disease, CRP is an interesting target to use to
explain plasma protein synthesis with the new approach.
1D and 2D electrophoresis of plasma. 1D and 2D electrophoresis was performed on a plasma sample of a 60-year-old
volunteer. After identification of the band (1D) or spot (2D)
containing CRP, it was cut out of the gel and digested
according to the method of Shevchenko et al (14). The
resulting peptide fraction was separated in a 15 cm 3 150 mm
(i.d.) reversed phase column, operated at a flow rate of 1 mL/
min (19), using a methanol gradient and 0.1% formic acid
containing solvents on a chromatographic system, which is
essentially comparable to the system described by Meiring et al
(19). To study lower concentrations of proteins and/or proteins
with a molecular mass below 100,000 Da, the 1D approach is
usually the method of choice to identify and isolate target
proteins [for reference (20,21)].
ESI-MS of CRP spot. The column effluent was directed
into a Model LCQ-Classic ion-trap mass spectrometer equipped
with a dynamic nanospray probe (Thermo-Finnigan, Breda,
Netherlands). MS-MS spectra were collected from the eluting
peptides and used to confirm the identity of the CRP spot by
the amino acid sequence of the peptides. Next, a target peptide,
as specific as possible for CRP, was selected by theoretically
cutting the CRP molecule by the action of trypsin using a free
available peptide cutting program (22). The presence of this
peptide in the actual digest was confirmed on the basis of its
molecular mass and by using MS-MS fragmentation, confirming
the peptide sequence true the presence of the complete B and Y
ion set (Fig. 1). In a second run, its retention time in the
chromatogram was established while at the same time the MS
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SUPPLEMENT
FIGURE 1 MS-MS spectrum of peptide Ala-Phe-Val-Phe-Pro-Val (AFVFPV) derived from a tryptic digest of CRP confirming its identity. Identified B
(AF, AFV, AFVF and AFVFP) ions and Y (PV, FPV, VFPV and FVFPV) ions are indicated in the figure.
system was set to a higher resolution mode called zoom-scan
enabling the baseline separation of the isotopomers of the target
peptide (Fig. 2). In this situation, the isotopic enrichment of
the peptide was determined and used to calculate the synthetic
rate.
Posttranslational modifications. In addition to the abovedescribed procedure the collected MS-MS scans can be used for
another purpose. The fragmentation patterns of the peptides
reveal not only their sequence, but can also be used to establish
the presence of a posttranslational modification (9). To enable
this, a peptide fragment of the target protein must be identified,
which may or may not be posttranslationally modified and followed by a measurement of the enrichment after tracer administration. It must be considered that modification of the protein
FIGURE 2 The total ion current of peptide Ala-Phe-Val-Phe-Pro-Val (AFVFPV) at 40 min (Panel A) and zoom-scan spectrum of this peptide (Panel
B). From the data of the zoom-scan, the ion current of the different isotopomers is measured and used in the calculation of protein synthesis.
PLASMA PROTEIN SYNTHESIS USING PROTEOMICS
by itself may result in a different elution pattern in the preceding isolation procedure. As an example (Fig. 3), the chromatogram of the peptide: YLYEIAR at 29.4 min and the nitrosylated
form: nYLYEIAR at 35.5 min in the chromatogram of the tryptic
digest of nitrosylated bovine serum albumin is shown.
It is more appropriate to use, if possible, a one-step isolation
procedure like 1D electrophoresis or affinity chromatography,
which is likely to minimally affect the elution position of the
target protein. Using 2D electrophoresis it might be expected
that the modified protein elute at a different place in the gel
making it more complicated to identify.
Measurement of protein synthesis, using the
ESI-MS approach
Introduction to the principle. To be able to measure their
synthesis rate, proteins have to be labeled with isotopes. In
human studies, application of stable isotopes tracers is of course
preferable. Typically, a continuous infusion of a tracer like
L-[5,5,5-2H3]Leucine or L-[1-13C]Leucine is started after
a priming dose at a level to obtain a steady-state plasma
leucine enrichment of 5–10%. The advantage of leucine tracers
is their availability and low cost. The duration of the infusion
depends on the enrichment in the protein that is required for
reliable measurement in the peptide of interest, which is in turn
dependent on the sensitivity of the available MS system. The
disadvantage is that the target protein should contain a leucinecontaining peptide fragment, which is released through a tryptic
cleavage. In the case of CRP, this proved to be a problem. The
most consistent peptide obtained from a tryptic digest that was
sufficiently abundant was the peptide Ala-Phe-Val-Phe-Pro-
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Val. Therefore, in this particular situation we infused labeled
phenylalanine.
Example of measuring protein synthesis of CRP. To test
the validity of this approach we propose the following scheme.
A healthy human volunteer is infused with L-[2H3] leucine at
a rate of 2.5 mmol/kg bw/h after a priming dose of 2.2 mmol/kg
bw. We have determined that steady-state conditions for
plasma leucine in this protocol is obtained after 30–45 min (not
shown), whereas a continuous infusion for 6 h is required to
obtain sufficient enrichment. Plasma samples are obtained just
before start of the stable isotope infusion (for basal enrichment)
and at 6 h of infusion. 1D gel electrophoresis is done on the
plasma samples; from which the CRP-containing band is cut
out to make tryptic digests. The LC-MS should be set to zoomscan resolution to detect the CRP target peptide AFVFPV with
a molecular mass of ;708 (1H) to measure its isotopic
enrichment (Fig. 2). The accuracy of this approach in our
hands is determined by dividing a CRP standard into 25 ml
fractions at a physiological concentration (5 mg/L), followed by
digestion with trypsin and measurement of the isotopic ratios of
the CRP target peptide AFVFPV (Table 1).
How can plasma proteomics be used to assess
amino acid adequacy?
Several studies have used the measurement of plasma
protein synthesis as a method to determine the quality of
proteins. Studies from the group of Bernard Beaufrere (2) have
shown that the synthesis of plasma albumin is stimulated after
a meal and that this stimulation is related to the amino acid
composition of the protein ingested. Jackson et al (5) had
FIGURE 3 The relative abundance of the peptide: YLYEIAR (Tyr-Leu-Tyr-Glu-Ile-Ala-Arg) eluting at 29.4 min and the nitrosylated form: nYLYEIAR
eluting at 35.5 min in the chromatogram of the tryptic digest of nitrosylated bovine serum albumin (BSA) is shown. Assuming that the ionization efficiency is
equal between the peptides, their relative abundance can be used to calculate the amount of the nitrosylated form of the peptide to be ;18.8%.
Enrichment measurements of these peptides is performed by comparing the M 1 1, M 1 2, M 1 3, etc. masses above the base mass (M 1 0) of the
respective peptides.
SUPPLEMENT
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TABLE 1
Precision of CRP target peptide (AFVFPV) enrichment
measurement obtained in zoom-scan resolution (N 5 9) after
digestion of 25 ml aliquots at a 5 mg/L concentration
Ratio
M1/M0
M2/M0
M3/M0
M4/M0
Percentage
43.47
10.76
1.64
0.24
6
6
6
6
0.24
0.20
0.18
0.07
Results are expressed as the mean 6 SD percentage of the abundance
of the isotopomer (M1–M4) peaks from the nonenriched base peak (M0).
observed that reducing the protein intake differentially affects
the synthesis of plasma proteins.
In line with these results, it is anticipated that the synthesis
of many plasma proteins will respond to the amount and composition of the enteral nutrition. It is still unknown whether
plasma proteins besides albumin respond to nutrition and
whether their response in synthesis is related to the quality and
adequacy of protein and amino acids in the meal. It may well be
that there are plasma proteins that can be used as very sensitive
markers for the quality of the protein/amino acid meal. Further
studies are necessary to confirm this hypothesis.
In addition, studying tissue proteins is possible with the new
approach. However, this will be more difficult because the
number of tissue proteins is much larger and usually the
concentration of protein is lower.
How can plasma proteomics be used to assess amino
acid safety?
What are the posttranslational modifications of amino acids
in a protein? The occurrence of many types of posttranslational modifications of proteins has already been known for some
time (23). Furthermore, it is well known that this type of
modification of a protein can be related to disease, for example,
glycosylation of albumin as a result of the increased glucose level
existing in persons with diabetes mellitus (24,25). Although the
presence of such a modification is believed to influence protein
function, we postulated that it would also affect protein synthesis
and/or degradation rates. In this respect we thought it to be of
interest to establish if there is a difference between the synthetic
rate of a modified protein and that of a nonmodified protein. For
instance, modifications are known for plasma fibrinogen (26),
lipoproteins (27) and albumin (25). There are posttranslational
modifications of amino acids, such as modifying an amino acid
into another amino acid or modifications that change the redox
state of a protein (24). Well-known modifications are, besides
glycosylation, nitrosylation, phosphorylation and ubiquitination
(8), the methylation of histidine (3-methyl-histidine) in actin
and myosin, the hydroxylation of proline (hydroxyproline) and
the removal of a guanidino group from arginine to become
citrulline.
Are there posttranslational modifications induced by amino
acids? The administration of arginine in diseased animals and
humans is known to enhance nitric oxide synthesis (28,29).
Nitric oxide can be attached to tyrosine in protein resulting
in nitrosylation, a posttranslational modification (30,31) that
likely affects the function of the protein.
There are also posttranslational modifications in which an
amino acid is attached to an amino acid within the protein
(23). One example of this type is the well-known arginylation of
proteins (32,33), which involved the addition of arginine to the
N-terminal residues: aspartate, glutamate and cysteine (34).
Arginylation of proteins has a regulatory role in protein
breakdown by the ubiquitin route (33) and programmed cell
death (35). In addition, a regulatory role in cardiovascular
development has been suggested (34). Because arginylation
takes place intracellularly, it is probably not a common
modification of plasma proteins.
The regulation of these and many other posttranslational
modifications by amino acids is at the moment unknown.
This field of research deserves more attention to be able
to understand amino acid safety in relation to protein
function.
CONCLUSION
In conclusion, innovative proteomic techniques using high
performance MS will provide more information about plasma
protein synthesis. It enables simultaneous measurement of the
synthesis and posttranslational modifications of many plasma
proteins and its relation to nutrition and metabolism in health
and disease.
ACKNOWLEDGMENTS
The authors wish to thank Freek Bouwman and Johan Renes from
the Maastricht Proteomics Center for performing the electrophoresis.
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