Reliable and generic liquid chromatography/mass spectrometry quantification of short peptides using a stable-isotope-labeled labeling agent.

RATIONALE: It is important to investigate the behavior of protein hydrolysate components in both in vitro and in vivo studies, to support the elucidation of their biological functions. As protein hydrolysates and biological matrices are highly complex mixtures, it is essential to apply fully reliable and flexible analytical approaches. METHODS: A novel and generic Liquid Chromatography/Mass Spectrometry methodology was developed to analyze short peptides. A stable-isotope-labeled labeling agent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (13 C3 ) was synthesized and used to prepare internal standards from non-labeled analyte peptides. The amino acid and peptides p, pG, Pp, GPp and PpG (where p stands for hydroxyproline) were used for proof of principle. RESULTS: The method showed acceptable performance in solvent, in simulated gastrointestinal fluid and in serum. The (linear) dynamic range expanded to over four orders of magnitude, which is very useful when multiple analytes are analyzed in a biological matrix, due to the large differences in concentrations observed for endogenous and protein hydrolysate components. The method provides absolute-quantitative results and is fully accountable on the single-sample and single-component level. CONCLUSIONS: The methodology can be applied to reliably quantify protein hydrolysate nutraceutical components at various stages during their in vivo processing. Internal standards can also be synthesized for other short peptides whenever they are expected to have biological relevance and require quantification. Overall this provides an excellent analytical tool to support the elucidation of the biological functions of protein hydrolysate components.

Non-targeted and targeted analysis of collagen hydrolysates during the course of digestion and absorption.

Protein hydrolysates are an important part of the human diet. Often, they are prepared from milk, soy, or collagen. In the present study, four different collagen hydrolysates were tested, varying in the average molecular weight and the animal source. Three types of samples, the dissolved start products, in vitro generated dialysates (containing the digested components that are potentially available for small intestinal absorption), and human serum collected after product ingestion, were analyzed using LC-MS to compare the state of the hydrolysates before and after absorption, i.e., uptake into the blood. It was found that the composition of the collagen hydrolysates prior to and after ingestion was highly complex and dynamic, which made it challenging to predefine a strategy for a targeted analysis. Therefore, we implemented a new analytical approach to first map hydrolysate data sets by performing non-targeted LC-MS analysis followed by non-targeted and targeted data analysis. It was shown that the insight gained by following such a top down (data) analytical workflow could be crucial for defining a suitable targeted setup and considering data trends beyond the defined targets. After having defined and performed a limited targeted analysis, it was found that, in our experimental setup, Hyp-Gly and especially Pro-Hyp contributed significantly as carrier to the total Hyp increase in blood after ingestion of collagen hydrolysate. Graphical abstract.

Domain-Specific Proteogenomic Analysis of Collagens to Evaluate De Novo Sequencing Results and Database Information.

Collagen is an important structural protein and the most abundant protein in mammals. In several research fields, structural analysis of collagens is performed. Fibrillar collagens almost entirely consist of continuous repeats of GXY, where G is glycine, X is often proline or alanine and Y is often hydroxyproline or alanine. In the present study, the collagen structure was investigated in detail at the nucleotide, codon group, amino acid and target peptide level using sequence analyses. One of the most important findings was that a selection of codon groups is predominantly involved in amino acid changes between closely related collagens and that other change routes come up when collagens are less related. The findings of the sequence analyses were used to evaluate reported sequences of non-avian dinosaur species and database entries of duck and chicken collagen. The duck assessment was supported by an experimental data set, obtained by collagen extraction from duck skin and subsequent digestion and LC-MS analysis. It was found that database entries of chicken and duck collagen 3α1 contained unreliable features, such as missing parts, no continuous GXY pattern and too many interspecies differences. As an example, the erroneous nature of one of these unreliable features was confirmed experimentally using LC-MS. Finally, dino and bird collagen 1α1 were compared. The presented results will show that performing a domain-specific proteogenomic analysis provides very useful information to assess de novo sequencing results and database information of collagens. Furthermore, it offers deeper insight in the functional restrictions and routes of evolutionary divergence.

Convergent analysis of food products using molecular barcodes, based on LC-HRMS data.

There are various analytical techniques available to address the growing interest in the composition of food products. LC-HRMS(/MS) is the most comprehensive technique, providing detailed information at the molecular level. However, given the vast number of different molecules encountered in food products, it is important to obtain a global overview of the dataset before focusing on similarities and differences. Therefore, a convergent strategy was employed, going from non-targeted to targeted analysis, with insightful data representations, most notably Molecular Barcode. Additionally an intermediate, semi-targeted analysis was defined, aimed at the specific detection of animal tissue in food products, using pG+ and related fragments after all ion fragmentation. The use of Molecular Barcode as a starting point to obtain relevant molecular data was also demonstrated. In conclusion, the convergent approach facilitates the design of suitable targeted methods, either to discriminate between samples or to find a generic target.

The quest for a generic bird target to detect the presence of bird in food products and considerations for paleoprotein analysis.

It can be important for consumers to know whether food products contain animal material and, if so, of which species. Food products with animal material as an ingredient often contain collagen type 1. LC-MS/MS (Liquid Chromatography-tandem Mass Spectrometry) was applied as technique to generically detect bird. Unlike for example fish, that have experienced longer divergence times, it is still possible to find generic LC-MS targets for avian type 1 collagen. After theoretical target selection using 83 collagen 1α2 bird sequences of 33 orders and construction of a common ancestor sequence of birds, experimental evidence was provided by analyzing extracts from 10 extant bird species. Two suitable options have been identified. The combination of VGPIGPAGNR and VGPIGAAGNR (pheasant only) covers all investigated birds and was not found in other species. The peptide EGPVGFpGADGR covers all investigated birds, but also occurs in several species of crocodiles and turtles. The presence of the generic peptide (combination) was confirmed in food products, proving the principle, and can therefore be used to detect the presence of bird. Furthermore, it is shown how the use of constructed ancestor sequences could benefit the field of paleoproteomics, in the interpretation of collagen MS/MS spectra of ancient species. Our theoretical analysis and assessment of reported Brachylophosaurus canadensis collagen 1α2 MS/MS data provided support for several previous peptide sequence assignments, but we also propose that our constructed ancestral bird sequence GPpGESGAVGPAGPIGSR may fit the MS/MS data better than the original assignment GLPGESGAVGPAGPpGSR.

Identification of collagen 1α3 in teleost fish species and typical collision induced internal fragmentations.

In contrast to collagens 1α1 and 1α2, the more obscure collagen 1α3 is sparsely mentioned in literature. In skin collagen type 1 of teleosts (bony fish), however, the chain occurs in a heterotrimer together with collagens 1α1 and 1α2, which makes it one of the most abundant proteins in teleosts. As teleost fish species and gelatin (hydrolysate) prepared from their skin are a major source for food products and nutraceuticals, the goal of the study was to selectively identify collagen 1α3 in several fish species. Fish skin extracts and fish skin gelatins were analyzed using LC-MS. Depending on the amount of available genetic information different approaches were used to identify collagen 1α3. Additionally, collagen-specific collision induced internal fragmentations are discussed, which are important to consider during data analysis. Ultimately the presence of collagen 1α3 could be confirmed using LC-MS in multiple fish species.

Integrated hemolysis monitoring for bottom-up protein bioanalysis.

Background: Hemolysis can result in analyte suppression or enhancement and it can affect the extraction efficiency and analyte stability. Triskelion developed an LC-MS method to monitor hemolysis. The concept can be integrated into existing and new quantitative protein LC-MS methods and can be validated according to the most appropriate tier. Results/methodology: In this proof of concept study, the tryptic target LLVVYPWTQR was used to quantify hemoglobin. The peptide target has only few variations considering the most common (laboratory) animals and is thus nearly generic. It was shown that LC-MS is a suitable technique for the quantification of hemoglobin in hemolyzed samples and that the signals are not affected by lipemia. Conclusion: LC-MS exhibited the best performance to monitor hemolysis when the results were compared with UV-VIS and visual inspection, especially when samples were lipemic.

Comprehensive two-dimensional liquid chromatography with ultraviolet, evaporative light scattering and mass spectrometric detection of triacylglycerols in corn oil.

An improved comprehensive two-dimensional (LC x LC) HPLC system for the analysis of triacylglycerols was developed. In the first-dimension, a Ag(I)-coated cation exchanger (250 mm x 2.1 mm, 5 microm) was employed with a gradient from 100% MeOH to 6% MeCN in MeOH at 20 microL/min. Using a 10-way valve with two switching loops, 1 min sections of the first-dimension were introduced in the second-dimension consisting of a 30 mm x 4.6 mm C18 (1.8 microm) column with an isocratic mobile phase of methanol-methyl tert-butyl ether (70:30) at 3.0 mL/min. As the second-dimension solvent was stronger than the first-dimension solvent, focusing in the second-dimension took place, leading to better separations than in previously reported analyses in which hexane was the main constituent of the first-dimension eluent. Compounds differing by 2 in their partition number were baseline separated in the second-dimension. Detection took place by UV at 210 nm, evaporative light scattering and (+)-atmospheric pressure chemical ionisation-MS with the latter giving the best results. Corn oil was investigated and 44 compounds could be detected: 34 triacylglycerols (TAGs), 8 oxygenated TAGs, and 2 TAGs containing a trans double bond. Data manipulation allowed the construction of contour plots and the automated calculation of the first- and second-dimension retention times and peak areas. Quantitative results are compared with a fatty acid methyl ester analysis, and with literature data.

Validation and theoretical justification of an LC-MS method for the animal species specific detection of gelatin.

Collagen is the most abundant protein family in mammals. Commercial edible gelatins are often produced from bovine and porcine skin and bone and consist mainly of partially hydrolyzed collagen type 1. The gelatin industry would benefit from a sensitive and reliable species detection method to unambiguously demonstrate species authenticity of their products. PCR and ELISA could in principle be used for this purpose. However, for gelatin, problems associated with false-positive and false-negative results, inconsistencies and low reactivity of commercially available kits have been observed with regard to ELISA and PCR methods. Therefore we developed a selective bottom-up LC-MS methodology for quantitative gelatin species determination with a lower limit of quantification of 0.05%. The present article describes the validation of this method, which was performed according to Good Laboratory Practice, and the theoretical justification for bovine and porcine target selection. The validated method can be used to determine the purity of gelatin batches with regard to bovine and porcine constituents.

Quantitative bottom up analysis of infliximab in serum using protein A purification and integrated µLC-electrospray chip IonKey MS/MS technology.

BACKGROUND: TNO Triskelion has applied its general workflow for the development of quantitative LC-MS methods for proteins in biological matrices to the quantification of infliximab in rat serum using bottom up µLC-MS/MS. Results/methodology: The general workflow consists of sample purification, analyte processing and LC-MS analysis. In the development of a quantitative µLC-MS/MS method for infliximab in rat serum the analyte processing part and the LC-MS part were optimized, in order to meet the different sample requirements of µLC-MS as compared with UPLC-MS. Using the optimized µLC-MS/MS method the LOQ was 75 ng/ml. CONCLUSION: The present study showed that it is possible to gain sensitivity when going to smaller scale LC-MS (UPLC-MS to µLC-MS). Due to the combination of a modified sample preparation approach and the application of µLC-MS a lower LOQ could be achieved for infliximab compared with a previously developed UPLC-MS method.

A generic method for the quantitative analysis of aminoglycosides (and spectinomycin) in animal tissue using methylated internal standards and liquid chromatography tandem mass spectrometry.

Aminoglycosides (AGs) are a large and diverse group of antibiotics. Although AGs may cause side effects of nephrotoxicity and ototoxicity, they are still occasionally being used for the treatment of serious infections. In this study the development of a method is described for the quantitative determination and confirmation of seven aminoglycosides (and relevant isomers) and spectinomycin in animal tissues. The extraction was based on an extraction followed by a concentration and clean-up step using weak cation exchange solid phase extraction. The separation was performed by ion-pair liquid chromatography on a C(18) column followed by mass spectrometric detection. The method was validated according to the EU requirements for a quantitative confirmatory method. Permethylated aminoglycosides (in-house synthesised internal standards) were used for accurate quantification. The accuracy of the analyses of AGs in kidney ranged from 94 to 111%, intra-day precision ranged between 2.5 and 7.4% (R.S.D.(r)) and inter-day precision ranged between 2.2 and 17.3% (R.S.D.(RL), n=21, MRL level). Accuracy (muscle tissue) varied from 83 to 128% with an intra-day precision between 2.2 and 17.3% (R.S.D.(r), n=7, MRL level). From the results it was concluded that the method was able to monitor MRL levels which ranged from 750 to 20,000 microgkg(-1) for kidney and from 50 to 10,000 microgkg(-1) for muscle tissue.