Mass Spectrometry Equipment Research Articles

Use of cestodes as indicator of heavy-metal pollution

Thirty snakehead fish, Channa micropeltes (Cuvier, 1831) were collected at Lake Kenyir, Malaysia. Muscle, liver, intestine and kidney tissues were removed from each fish and the intestine was opened to reveal cestodes. In order to assess the concentration of heavy metal in the environment, samples of water in the surface layer and sediment were also collected. Tissues were digested and the concentrations of manganese (Mn), zinc (Zn), copper (Cu), cadmium (Cd) and lead (Pb) were analysed by using inductively-coupled plasma mass-spectrometry (ICP-MS) equipment. The results demonstrated that the cestode Senga parva (Fernando and Furtado, 1964) from fish hosts accumulated some heavy metals to a greater extent than the water and some fish tissues, but less than the sediment. In three (Pb, Zn and Mn) of the five elements measured, cestodes accumulated the highest metal concentrations, and in remaining two (Cu and Cd), the second highest metal accumulation was recorded in the cestodes when compared to host tissues. Therefore, the present study indicated that Senga parva accumulated metals and might have potential as a bioindicator of heavy-metal pollution.

Quantitative analysis of amino acids in physiological fluids by UPLC-MS/MS with stable isotope internal standards

Amino acid analysis is an essential service for the diagnosis and management of inborn errors of metabolism (IEM). For over 50 years the gold standard technique has been lithium ion exchange chromatography (IEC) with post-column ninhydrin derivatisation and photometric detection. This technique is laborious and expensive with typical run times over 120 minutes and requires dedicated instrumentation. Reverse phase chromatography with pre-column derivatisation and either fluorimetric or photometric detection reduces runs times but can suffer from interference. Many biochemical genetics laboratories already have tandem mass spectrometry (MS/MS) equipment which can be linked to ultra performance liquid chromatography (UPLC). The combination of these two techniques gives the opportunity to produce highly specific quantitative assays with high throughput and we report on our experience with a UPLC-MS/MS system for amino acid analysis in physiological fluids. Twenty-three physiological amino acids and 13 stable isotope internal standards, using 34 mass pairs are analysed within a 20 minute run utilising a simple acetonitrile/water gradient with tridecafluoroheptanoic acid ion pairing reagent. Sample preparation is limited to ultrafiltration to remove protein and dilution with internal standard mixture in mobile phase. The method correlates well with IEC results and gives comparable precision and external QAP performance.

Identification and quantification of flavonol glycosides in cultivated blueberry cultivars

The aim of this work was to characterise native flavonol profiles of different varieties of blueberries during different growing seasons. The analysis was performed using liquid chromatography–mass spectrometry (LC/MS) equipment, with a reversed phase column for flavonol separation. The identity of blueberry flavonols was also confirmed using high-resolution and high-accuracy mass spectrometry. Relatively large differences between different varieties of blueberries were found in terms of their flavonol content. The sum of flavonol glycosides varied between 137mg/kg for the Duke variety to 272mg/kg for the Simultan variety. Quercetin was the most abundant in all varieties (59.4%±8.7%), while the sequence in terms of the presence of other flavonols was variety-dependent (from 16.3% to 1.6%). The most significant conjugate sugars in blueberry extracts were galactoside (ranging from 35.8% to 72.1%) and glucoside (ranging from 12.1% to 27.1%). The study demonstrated that the percentage of different sugar glycosides depends on the variety. These data support the conclusion that cultivated blueberries are one of the most concentrated sources of dietary flavonol glycosides in the human diet. They can provide on average 196mg/kg of a complex mixture of up to 23 different flavonol glycosides.

Modification of citrulline residues with 2,3‐butanedione facilitates their detection by liquid chromatography/mass spectrometry

Citrullination is a post-translational modification (PTM) that results from the deimination of the amino acid arginine into citrulline by Peptidyl Arginine Deiminase enzymes and occurs in a wide range of proteins in health and disease. This modification causes a 1 Da mass shift, which can be used to identify citrullination sites in proteins by the use of mass spectrometry. However, other PTMs, such as deamidation from asparagine to aspartic acid or from glutamine to glutamic acid, can also cause a 1 Da mass shift, making correct interpretation of the data more difficult. We developed a chemical tagging strategy which, combined with an open source search application, allowed us to selectively pinpoint citrullinated peptides in a complex mixture after liquid chromatography/mass spectrometry (LC/MS) analysis. After incubation of a peptide mixture with 2,3 butanedione, citrulline residues were covalently modified which resulted in a 50 Da shift in singly charged mass. By comparison of the peptide mass fingerprint from a modified and an unmodified version of the same sample, our in-house search application was able to identify the citrullinated peptides in the mixture. This strategy was optimized on synthetic peptides and validated on a digest of in vitro citrullinated fibrinogen, where different proteolytic enzymes were used to augment the protein coverage. This new method results in easy detection of citrullinated residues, without the need for complex mass spectrometry equipment.

Using the Mini-Session Course Format To Train Students in the Practical Aspects of Modern Mass Spectrometry

Mass spectrometry (MS) has been gaining in popularity in recent years owing in large part to the development of soft-ionization techniques such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). These soft-ionization techniques have opened up the field of MS analysis to biomolecules, polymers, and other high molecular weight materials. The growing publication rate and a quick survey of available job postings requiring MS skill sets illustrate the importance of properly training students in the use of modern MS techniques. Many colleges and universities are unable to hire full-time technical staff devoted to student training on the use of MS equipment. The lack of technical staff results in less than adequate student training on the use of MS equipment, which ultimately leads to student apprehension about using the instruments regularly in their research projects. Herein, we report on our efforts utilizing a mini-session course format (one week with 40 contact hours) to properly train graduate students in the use of modern MS equipment and techniques. The course consists of three components: (i) a lecture component of approximately 2 h each day to introduce the basic theory of the days topics, (ii) a hands-on laboratory component illustrating the use of both MALDI and ESI instruments to gather a variety of data, and (iii) a student project utilizing the MS equipment in their own graduate research during the regular semester following the mini-session course. The students receive three hours of graduate credit and receive permission, upon successful completion of the course, to use the MS facilities unsupervised. The faculty member teaching the course receives teaching credit for the training efforts.

A Proteomic-Based Approach for Detection of Chicken in Meat Mixes

A proteomic-based method has been developed for the detection of chicken meat within mixed meat preparations. The procedure is robust and simple, comprising the extraction of myofibrillar proteins, enrichment of target proteins using OFFGEL isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Using this approach, it was possible for example to detect 0.5% contaminating chicken in pork meat with high confidence. Quantitative detection of chicken meat was done by using AQUA stable isotope peptides made from the sequence of previously selected species-specific peptide biomarkers. Linearity was observed between the amount of the peptide biomarker and the amount of chicken present in the mixture; further independent replication is required now to validate the method. Apart from its simplicity, this approach has the advantage that it can be used effectively for the detection of both raw and cooked meat. The method is robust, reliable, and sensitive, representing a serious alternative to methods currently in use for these purposes. It is amenable to highly processed foods which can be particularly problematic, as the tertiary protein structure is often affected in processed food precluding immunoassays. In addition, this proteomic analysis will permit the determination of definitive discriminatory sequence, unlike the DNA PCR based methods used presently. The present article also demonstrates the translation of the technology to routine mass spectrometry equipment, making the methodology suitable for public analysts.

On the acquisition of +1 charge states during high-throughput proteomics: Implications on reproducibility, number and confidence of protein identifications

Modern high-throughput methods for the proteome analysis are gradually replacing more traditional 2D gel-based techniques. Almost immediately after the introduction of high-throughput proteomics techniques in 2001, reproducibility of the results became an issue. Extensive discussion in the literature led to the conclusion that certain "undersampling" exhibited during measurements could be due to the stochastic nature of the data-dependent sampling, routinely used with current mass spectrometry equipment. At the same time, the effect of the acquisition of different charge states on the reproducibility and confidence of protein identifications, to the best of our knowledge, has never been properly evaluated. There exists the frequently voiced yet hardly documented opinion that +1 charge states should be rejected during data-dependent acquisition. The work presented here shows that inclusion of the +1 charge state in the data-dependent acquisition protocols can indeed lead to improved proteome coverage, reproducibility, and the confidence of protein identifications by high-throughput proteomics. It was also shown that contrary to the established opinion, gas-phase dissociation of singly charged peptide species results in rich fragmentation patterns containing both b- and y-ions allowing for successful and confident peptide identification.

Fully Automatic Sample Treatment by Integration of Microextraction by Packed Sorbents into Commercial Capillary Electrophoresis−Mass Spectrometry Equipment: Application to the Determination of Fluoroquinolones in Urine

This paper describes a new and innovative way to integrate microextraction by packed sorbents (MEPS) into commercial CE equipment. The suggested integration allows the automatic sample cleanup and preconcentration requiring only a few microliters of sample and no additional hardware and software. The MEPS was integrated in the outlet region of a commercial CE equipment cartridge in order to provide easy manipulation and exchange. The robustness of the proposed integration was demonstrated by the design and use of a (MEPS)-nonaqueous capillary electrophoresis (NACE)-MS method used to determine fluoroquinolones "FQs" (namely, ofloxacin, marbofloxacin, enrofloxacin, danofloxacin, and difloxacin) in urine. The method allows the analysis of micrograms per liter of FQs to be carried out with only 48 microL of urine sample. The obtained LODs were in the range 6.3-10.6 microg/L. An analysis of spiked urine samples was used to validate the method. Absolute recoveries were in the range of 71-109% while the precision expressed as repetitivity of peak area was lower than 5.9%.

Methods for Preparing Phenolic Extracts from Olive Cake for Potential Application as Food Antioxidants

Olive cake, the most important byproduct of olive oil extraction by the two-phase centrifugation system, was used to obtain phenolic extracts. The extracts were obtained using the two constituents of this waste, vegetative water and solid residue, to maximize the extraction of all phenolic compounds. Different extraction procedures were studied, a simple and rapid extraction procedure being developed from the solid residue using an accelerated solvent extractor (ASE). Afterward, the phenolic extracts were fractionated using semipreparative HPLC to study the antioxidant activity of the different components. The identification of the phenolic compounds was carried out with an ultraperformance liquid chromatograph coupled to tandem mass spectrometry equipment (UPLC-ESI-MS/MS). With this method, a complete list of the polyphenols from the extract was obtained. Finally, the antioxidant activity of the phenolic extracts and the isolated fractions was evaluated, showing great antioxidant capacities, between 3450 and 17900 micromol of Trolox equivalents/g of extract. With regard to the isolated fractions the most antioxidant were those that contained hydroxytyrosol and 3,4-DHPEA-EDA. The suitability of the solid residue extract obtained by the ASE procedure was demonstrated given the great range of phenolic compounds and the feasibility of production on an industrial scale.

Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high-performance liquid chromatography and electrospray ionization mass spectrometry

We examined the suppression methodology of the interaction between phosphate compounds, such as nucleotides, and the stainless steel surfaces of high-performance liquid chromatography and electrospray ionization mass spectrometry (HPLC/ESI-MS) equipment in an effort to prevent the tailing of peaks seen in HPLC chromatograms of phosphate compounds. Addition of carbonate (CO 3 2−) to mobile phase was highly effective in suppressing the interaction of phosphate compounds derived from a complexation between phosphate groups and metal ions that exist on a stainless steel surface in a mechanism similar to Fe(III)- and Cr(III)-immobilized metal affinity chromatography (IMAC). Addition of ammonium hydrogen carbonate to mobile phase achieved a simple and reliable HPLC/ESI-MS analysis of mono-, di-, and triphosphate compounds (six nucleotides) without peak tailing due to the interaction between stainless steel surfaces and phosphate groups. Moreover, ammonium hydrogen carbonate buffer, a volatile buffer with good buffering capacity at neutral pH, does not compromise the stability of silica-based HPLC columns, decreases in sensitivity, ion source pollution, clogging of the ESI interface, and/or ion suppression in HPLC/ESI-MS.

Influence of ZrO 2 in HfO 2 on reflectance of HfO 2/SiO 2 multilayer at 248 nm prepared by electron-beam evaporation

Influence of ZrO 2 in HfO 2 on the reflectance of HfO 2/SiO 2 multilayer at 248 nm was investigated. Two kinds of HfO 2 with different ZrO 2 content were chosen as high refractive index material and the same kind of SiO 2 as low refractive index material to prepare the mirrors by electron-beam evaporation. The impurities in two kinds of HfO 2 starting coating materials and in their corresponding single layer thin films were determined through glow discharge mass spectrum (GDMS) technology and secondary ion mass spectrometry (SIMS) equipment, respectively. It showed that between the two kinds of HfO 2, either the bulk materials or their corresponding films, the difference of ZrO 2 was much larger than that of the other impurities such as Ti and Fe. It is the Zr element that affects the property of thin films. Both in theoretical and in experimental, the mirror prepared with the HfO 2 starting material containing more Zr content has a lower reflectance. Because the extinction coefficient of zirconia is relatively high in UV region, it can be treated as one kind of absorbing defects to influence the optical property of the mirrors.

HfO2的纯度对紫外多层膜反射率的影响

The impurities in two kinds of HfO2 materials and in their corresponding single layer thin films were determined through glow discharge mass spectrum technology and secondary ion mass spectrometry (SIMS) equipment respectively. It was found that ZrO2 was the main impurity in the two kinds of HfO2 either in the original HfO2 materials or in the electron beam deposited films. In addition, the difference of Zr content in the two kinds of HfO2 single layer films was much larger than that of the other impurities such as Ti and Fe, which showed that it was just ZrO2 that made the difference between the optical performance of the film products including the two kinds of HfO2. With these two kinds of HfO2 and the same kind of SiO2, we deposited HfO2/SiO2 multilayer reflective coatings at the wavelength of 266 nm. Experimental results showed that the reflectances of these two mirrors were about 99.85% and 99.15% respectively, which agreed well with the designed results what were based on the optical constants obtained from the corresponding single layer thin films.

Alternatives for coupling sequential injection systems to commercial capillary electrophoresis–mass spectrometry equipment

On-line coupling of an automated flow system with a commercially available capillary electrophoresis (CE) system with an electrospray interface (ESI) for mass spectroscopic (MS) detection is described. The peculiarities of CE–ESI-MS interfaces, in which a high electrical field must be applied to the capillary end where the sample is provided by the flow system, introduce significant difficulties for the appropriate work of the entire arrangement. Experimental strategies are proposed for achieving stable conditions for on-line sample pre-treatment, conditioning of the separation capillary, sample injection, as the proper separation. The versatility and robustness of the proposed arrangement is discussed, taken as example the separation of a variety of amines. Connection of the CE system's pressure to the automated flow system enables hydrodynamic introduction of sample with high precision. The developed hyphenated system is of practical relevance as it opens an avenue for the simplification and automation of the whole analytical process required when using powerful CE–ESI-MS equipments.

Identification of human nasal mucous proteins using proteomics

The determination of possible biomarkers in nasal secretion of healthy subjects can have a role in early diagnosis of diseases such as rhinosinusitis. For this purpose, nasal lavage fluids (NLFs) from ten volunteers, collected before and after they had been submitted to nasal provocations, were investigated. Separation and analysis of proteins present in this complex matrix was performed using a capillary liquid chromatography-electrospray-quadrupole-time of flight mass spectrometry equipment. From among a total of 111 proteins found (89 known and two unknown proteins), 42 of which had never been previously described in this fluid, such as Deleted in Malignant Brain Tumors 1 isoform a precursors, and cytoskeletal proteins were identified with high statistical score. Three proteins of palate lung nasal epithelial clone (PLUNC) family: SPLUNC1, LPLUNC1, and LPLUNC2 were identified. Proteins involved in innate (27%) and acquired immunity (21%) systems were major components of NLF. Cellular (52% of all proteins identified) such as cytoskeletal (33%), functional (15%), and regulatory (4%) proteins, normally present in the nasal cavity, have also been identified. The proteomic approach presented here allowed us to identify the proteins involved in acquired and innate immune response in the nose against microbial infections and unclean inhaled air.

Analytical error in stable isotope ecology

The increasing popularity of stable isotope analysis (SIA) as an ecological research tool and the ease of automated analysis have created a knowledge gap between ecologists using SIA and the operators of isotope ratio mass spectrometry (IRMS) equipment. This has led to deterioration in the understanding of IRMS methodology and its proper dissemination in the ecological literature. Of 330 ecological research papers surveyed, 63 (19%) failed to report any form of analytical error associated with IRMS. Of the 267 papers that reported analytical error, there was considerable variation both in the terminology and approach used to quantify and describe error. Internal laboratory standards were often used to determine the analytical error associated with IRMS, so chosen because they are homogenous and have isotopic signatures that do not vary over time. We argue that true ecological samples collected in the field are complex bulk mixtures and often fail to adhere to these two criteria. Hence the analytical error associated with samples is potentially greater than that of standards. A set of standard data run over time with a precision typically reported in the ecological literature (1 standard deviation: 1SD = 0.26 per thousand) was simulated to determine the likelihood of spurious treatment effects depending on timing of analysis. There was a 90% likelihood of detecting a significant difference in the stable nitrogen ratio of a single sample (homogenized bovine liver) run in two time periods when n > 30. Minor protocol adjustments, including the submission of blind replicates by researchers, random assignment of sample repeats within a run by analytical labs, and reporting 1SD of a single sample analyzed both within and between runs, will only serve to strengthen the interpretation of true ecological processes by both researchers and reviewers.

Quantify this! Report on a round table discussion on quantitative mass spectrometry in proteomics.

Following the success of the first round table in 2001, the Swiss Proteomic Society has organized two additional specific events during its last two meetings: a proteomic application exercise in 2002 and a round table in 2003. Such events have as their main objective to bring together, around a challenging topic in mass spectrometry, two groups of specialists, those who develop and commercialize mass spectrometry equipment and software, and expert MS users for peptidomics and proteomics studies. The first round table (Geneva, 2001) entitled "Challenges in Mass Spectrometry" was supported by brief oral presentations that stressed critical questions in the field of MS development or applications (Stöcklin and Binz, Proteomics 2002, 2, 825-827). Topics such as (i) direct analysis of complex biological samples, (ii) status and perspectives for MS investigations of noncovalent peptide-ligant interactions; (iii) is it more appropriate to have complementary instruments rather than a universal equipment, (iv) standardization and improvement of the MS signals for protein identification, (v) what would be the new generation of equipment and finally (vi) how to keep hardware and software adapted to MS up-to-date and accessible to all. For the SPS'02 meeting (Lausanne, 2002), a full session alternative event "Proteomic Application Exercise" was proposed. Two different samples were prepared and sent to the different participants: 100 micro g of snake venom (a complex mixture of peptides and proteins) and 10-20 micro g of almost pure recombinant polypeptide derived from the shrimp Penaeus vannamei carrying an heterogeneous post-translational modification (PTM). Among the 15 participants that received the samples blind, eight returned results and most of them were asked to present their results emphasizing the strategy, the manpower and the instrumentation used during the congress (Binz et. al., Proteomics 2003, 3, 1562-1566). It appeared that for the snake venom extract, the quality of the results was not particularly dependant on the strategy used, as all approaches allowed Lication of identification of a certain number of protein families. The genus of the snake was identified in most cases, but the species was ambiguous. Surprisingly, the precise identification of the recombinant almost pure polypeptides appeared to be much more complicated than expected as only one group reported the full sequence. Finally the SPS'03 meeting reported here included a round table on the difficult and challenging task of "Quantification by Mass Spectrometry", a discussion sustained by four selected oral presentations on the use of stable isotopes, electrospray ionization versus matrix-assisted laser desorption/ionization approaches to quantify peptides and proteins in biological fluids, the handling of differential two-dimensional liquid chromatography tandem mass spectrometry data resulting from high throughput experiments, and the quantitative analysis of PTMs. During these three events at the SPS meetings, the impressive quality and quantity of exchanges between the developers and providers of mass spectrometry equipment and software, expert users and the audience, were a key element for the success of these fruitful events and will have definitively paved the way for future round tables and challenging exercises at SPS meetings.

Accelerator Mass Spectrometry at High Voltage Engineering Europa (HVEE)

In recent years, High Voltage Engineering Europa (HVEE) has demonstrated its capability of developing and installing turnkey accelerator mass spectrometry (AMS) equipment for the analysis of, among others, 14C and 129I. Five 3MV systems using sequential and/or simultaneous injection are operational in the field today, and they have shown excellent long-term stability providing high-precision analyses on a routine basis. Another three AMS systems are in production. It is projected that for the time being, AMS will continue to broaden its field of applications; a saturation of the market of AMS facilities is not expected in the near future.To meet the specific demands of the biomedical research community, we have developed an extreme compact 14C AMS system comprising a hybrid ion source capable of handling both graphite as well as CO2 samples. The source is optimized for easy maintenance, accommodates up to 200 samples, and can be implemented in any other AMS system.More recently, HVEE has initiated the development of an AMS system capable of detecting, among others, 36Cl and 41Ca. The design will include a HVEE 5MV Tandetron™. The accelerator is currently under construction as part of an ion beam analysis system for the Universidad Autonoma de Madrid (Spain).

The identification and characterization of silicate complexes in calcium chloride solution using fast atom bombardment mass spectrometry

The chemical species of silica dissolved in calcium chloride (CaCl 2) solution were identified using fast atom bombardment mass spectrometry (negative ion mode) equipment. The chemical species dissolved in CaCl 2 solution were compared with those in the sodium chloride (NaCl) solution to discuss the stability of the complex in CaCl 2 solution. In 0.1 mol dm −3 (M) CaCl 2 solution, some new species of silica complexes were identified. The chemical species of silica dissolved in CaCl 2 solution were as follows: silica monomer, Si(OH) 3O − and Si(OH)O 3Ca −, the dimer species such as Si 2(OH) 5O 2 −, Si 2(OH) 3O 4Ca −, the linear tetramer such as Si 4(OH) 9O 4 −, Si 4(OH) 7O 6Ca −, Si 4(OH) 5O 8Ca 2 −, Si 4(OH) 3O 10Ca 3 − and Si 4(OH)O 12Ca 4 −, and the cyclic tetramer such as Si 4(OH) 7O 5 −, Si 4(OH) 5O 7Ca −, Si 4(OH) 3O 9Ca 2 − and Si 4(OH)O 11Ca 3 −. The linear tetramer silica complexes were contained in the 0.1 M CaCl 2 solution, and the eight protons of the linear tetramer silicate Si 4(OH) 9O 4 − are all exchangeable with Ca 2+, while the linear tetramer silica complexes were not identified in 0.1 M NaCl solution. This indicates that the Ca-silicate complex is more stable than Na-silicate complex in each chloride solution, and that Ca 2+ should assist in the two intra-molecular bondings of Si-O −.

Use of pyrolysis–g.c.–m.s. to assess the thermal degradation behaviour of polymers containing chlorineI. The limits of detection and measurement of HCl, deduced from a study of PVC pyrolysis

From measurements of the yields of hydrogen chloride evolved from the thermal degradation of PVC samples, it is deduced that the authors' pyrolysis–gas chromatography–mass spectrometry equipment can detect HCl down to at least 50 ng (1.4 nanomoles). The actual limit is almost certainly smaller, because the calculations assume that HCl is obtained in quantitative yield from PVC on prolonged pyrolysis at 250°C. (Additional sensitivity may also be available by more specialised operation of the mass spectrometer in single ion monitoring mode, but the present work relates to the normal scanning mode used in general analysis). The above measured detection limit is more than 4000 times better than that obtained using g.c. equipment incorporating packed columns and a thermal conductivity detector, because with the latter apparatus there is substantial loss of HCl, probably due to reaction with the column packing. The prolysis–g.c.–m.s. results also provide an absolute sensitivity calibration for HCl, and in addition, permit calculation of the rate constant (specific rate, k) for HCl evolution from PVC at 250°C. The value of k, as calculated from the initial rate, is found to be ca. 5×10 −3 s −1 at 250°C, and is independent of sample size over the range 10–40 micrograms (corresponding to sample thickness 2.5–10 microns on the pyrolysis filament). For samples of 30–40 micrograms, the specific rate increases with conversion to a value in the final stages which is approximately double that of the initial value. This phenomenon is consistent with an autoaccelerative mechanism in which HCl production is promoted by HCl itself, with the residence time for HCl increasing with both the sample size and the degree of cross-linking of the residue.

Determination of protein synthesis in human rectal cancer in situ by continuous [1-13C]leucine infusion.

Previous studies on human colorectal tumor protein synthesis in situ relied on techniques that required intra- or perioperative sampling to obtain a sufficient biopsy size. The purpose of the present study was to develop a new technique by use of new mass spectrometry equipment [capillary gas chromatography (GC)-combustion isotope ratio mass spectrometry (IRMS)], which allows reduction of the necessary sampling size. Thereby, tumor sampling could be done via conventional rectosigmoidoscopy, excluding the need for further disturbing invasive measures. Fifteen postabsorptive patients with localized rectal cancer received a primed-constant infusion of [1-13C]leucine (0.16 mumol.kg-1.min-1 constant, 9.6 mumol/kg prime). Forceps biopsies were taken after 3 and 6 h. In five subjects, tumor tissue and normal mucosa were studied simultaneously. Determination of protein-bound leucine enrichment was done by GC-IRMS, and GC-quadrupole MS was used to determine tracer-to-tracee ratios (tracer/tracee) for free intracellular leucine. GC-MS data demonstrated achievement of a steady state in the precursor pool enrichment after 3 h of isotope infusion (tracer/tracee at 3 h: 6.34 +/- 0.46%, at 6 h: 6.58 +/- 0.38%). Calculation of tumor protein synthesis yielded a fractional synthetic rate (FSR) of 1.06 +/- 0.11%/h or 25.5 +/- 2.6%/day (range 12.0-37.1%/day). At any time, protein-bound leucine enrichment was significantly higher in tumor tissue than in normal mucosa of the same subject. However, protein synthetic rates were comparable (tumor: 1.09 +/- 0.20%/h, mucosa: 1.29 +/- 0.28%/h). Thus combined GC-combustion IRMS and GC-/quadrupole MS provide a simple, reliable, and minimally invasive method to determine tumor FSR in situ, thereby excluding interferences common to previous methods. Tumor and mucosa tissues are similar with respect to protein synthesis, but they apparently differ with respect to leucine extraction from the arterial blood.