2nd Amino Acid Workshop - Maastricht Proteomics Center

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1 Presented at the conference ''The Second Workshop on the Assessment of. Adequate ..... Kwon, Y. T., Kashina, A. S., Davydov, I. V., Hu, R. G., An, J. Y., Seo,.
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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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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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.

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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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