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.

Nutritional content, protein quantity, protein quality and carbon footprint of plant-based drinks and semi-skimmed milk in the Netherlands and Europe.

OBJECTIVE: To compare the nutritional composition of bovine milk and several plant-based drinks with a focus on protein and essential amino acid content and to determine the ratio of essential amino acids to greenhouse gas emission. DESIGN: Nutritional information on the label was extracted for semi-skimmed milk, soy, oat, almond, coconut and rice drink from the Innova database between January 2017 and March 2020 for the Netherlands, Belgium, Germany, Spain, Italy, and Sweden. Protein and amino acids were measured and carbon footprint was calculated for a selection of Dutch products. Protein quality was determined by calculating the contribution to the WHO essential amino acids requirements. SETTING: The bovine milk and plant-based drinks market in Netherlands, Belgium, Germany, Spain, Italy, and Sweden. PARTICIPATING PRODUCTS: Semi-skimmed bovine milk and soy-, oat-, almond-, coconut- and rice drink. RESULTS: Nutritional label information was collected for 399 products. Milk naturally contains many micronutrients, e.g. vitamin B2, B12, and calcium. Approximately 50% of the regular plant-based drinks was fortified with calcium, whereas the organic plant-based drinks were mostly unfortified. Protein quantity and quality were highest in milk. Soy drink had the best protein quality to carbon footprint ratio and milk came second. CONCLUSIONS: The nutrition - climate change balance presented in this study, is in line with previous literature, which shows that semi-skimmed bovine milk and fortified soy drink deserve a place in a sustainable diet.

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.

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.

Visualization of Genetic Drift Processes Using the Conserved Collagen 1α1 GXY Domain.

Speciation proceeds by the accumulation of DNA differences in time. The genetic code changes as a result of genetic drift and by selective pressure. In variable domains, exposure to high selective pressure obscures the view on background mutations. Therefore, we characterized and visualized background mutations using the highly conserved collagen 1α1 GXY domain. Typical change routes were identified and the data set showed several indications that changes in the collagen 1α1 GXY domain have taken place randomly within a functionally restricted space. The types of nucleotide and codon group differences are similar across the vertebrate subphylum and gradually become less functionally neutral with increasing distance between species, which offers the opportunity for rapid visualization of evolutionary relations using a single domain. It was concluded that the findings and approach of the study could be important for analytical method development in authenticity research, especially when conserved domains are targeted.

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.

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.

The determination of exogenous formaldehyde in blood of rats during and after inhalation exposure.

Formaldehyde (FA) is suspected of being associated with the development of leukemia. An inhalation experiment with FA was performed in rats to study whether FA can enter the blood and could thus cause systemic toxicity in remote tissues such as the bone marrow. Therefore, a sophisticated analytical method was developed to detect blood concentrations of FA during and after single 6-h exposure by inhalation. In order to differentiate between exogenous and endogenous FA the rats were exposed to stable isotope ((13)C) labeled FA by inhalation. During and after exposure of the rats to (13)C-FA their blood was analyzed to determine the ratio between labeled and natural FA in blood and the total blood concentration of FA. With respect to sensitivity, with the applied method exogenous (13)C-FA could have been detected in blood at a concentration approximately 1.5% of the endogenous FA blood concentration. Exogenous (13)C-FA was not detectable in the blood of rats either during or up to 30 min after the exposure. It was concluded that the inhalation of (13)C-FA at 10 ppm for 6h did not result in an increase of the total FA concentration in blood.

Identification of multiple post-translational modifications in the porcine brain specific p25alpha.

P25alpha is a protein normally expressed in oligodendrocytes and subcellular relocalization of p25alpha occurs in multiple system atrophy, Parkinson's disease and Lewy body dementia along with ectopic expression in neurons. Moreover, it accumulates in Lewy body inclusions with aggregated alpha-synuclein and is a potent stimulator of alpha-synuclein aggregation. P25alpha is a phosphoprotein and post-translational modifications (PTMs) may play a role in its disease-related abnormalities. To investigate the spectrum of PTMs on p25alpha we cloned porcine p25alpha and isolated the protein from porcine brain. Using several complementary tandem mass spectrometry techniques for peptide mass analysis and amino acid sequencing, a comprehensive analysis of the PTMs on porcine p25alpha was performed. It was found that porcine p25alpha is heavily modified with a variety of modifications: phosphorylation, di- and trimethylation, citrullination and a HexNAc group. The modifications are localized within p25alpha's unfolded terminal domains and suggest that their functional states are regulated. This comprehensive mapping of p25alpha's PTMs will form the basis for future functional studies and investigations of p25alpha's potential role as a biomarker.

Analysis of histidine phosphorylation using tandem MS and ion-electron reactions.

Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns.

Does double electron capture lead to the formation of biradicals? An ECD-SORI-CID study on lacticin 481.

We studied lacticin 481, a small lantibiotic with three lanthionine bridges, by electron capture dissociation (ECD) in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Following electron capture, very little fragmentation was observed, but species formed by nondissociative single and multiple electron capture were abundant. Ions formed by double electron capture were subjected to sustained off resonance irradiation collision induced dissociation (SORI-CID) to determine whether stable biradicals were formed. In the SORI-CID spectra of the ions formed by double electron capture, some, but minor, H* radical loss was observed, which was not observed at all for regularly protonated ions. A small part of the ions formed by double electron capture are thus long-lived biradicals. Apart from the observed H* loss, the SORI-CID spectra of ions that captured two electrons was similar to that of regularly protonated ions and quite different from the SORI-CID spectra of radical ions formed by single electron capture. This implies that recombination of the two radical sites is the dominant process in biradical lacticin 481 ions, at least on the time scale of our SORI-CID experiments.

Electron capture dissociation at low temperatures reveals selective dissociations.

Electron capture dissociation at 86 K of the linear peptide Substance P produced just two backbone fragments, whereas at room temperature eight backbone fragments were formed. Similarly, with the cyclic peptide gramicidin S, just one backbone fragment was formed at 86 K but five at room temperature. The observation that some backbone scissions are active and others inactive, when all involve NC(alpha) cleavages and have a high rate constant, indicates that the more specific fragments at low temperatures reflects the reduced conformation heterogeneity at low temperatures. This is supported by reduced or inactive hydrogen loss, a channel that has previously been shown to be affected by conformation. The conclusion that the ECD fragments are a snapshot of the conformational (intramolecular solvation shell) heterogeneity helps explain how the relative intensities of ECD fragments can be different on different instrument and highlights the common theme in methodologies used to increase sequence coverage, namely an increase in the conformational heterogeneity of the precursor ion population.

Localization of intramolecular monosulfide bridges in lantibiotics determined with electron capture induced dissociation.

Electron capture induced dissociation (ECD) and collisionally activated dissociation (CAD) experiments were performed on four lanthionine bridge-containing antibiotics. ECD of lantibiotics produced mainly c and z* ions, as has been observed previously with other peptides, but more interestingly, the less common c* and z ions were observed in abundance in the ECD spectra. These fragments specifically resulted from the cleavage of both a backbone amine bond and the thioether bond in a lanthionine bridge. ECD seemed to induce mainly cleavages near the lanthionine bridges. This fragmentation pattern indicates that lanthionine bridges play a key role in the selectivity of the ECD process. A new mechanism is postulated describing the formation of c* and z ions. Comparative low-energy CAD did not show such specificity. Nondissociative ECD products were quite abundant, suggesting that relatively stable double and triple radicals can be formed in the ECD process. Our results suggest that ECD can be used as a tool to identify the C-terminal attachment site of lanthionine bridges in newly discovered lantibiotics.