Species-specific Protein Research Articles

A rapid high-performance liquid chromatography-tandem mass spectrometry assay for unambiguous detection of different milk species employed in cheese manufacturing

The aim of the study was to investigate the possibility to differentiate the 4 most important species in Italian dairy industry (cow, buffalo, sheep, and goat), applying a bottom-up proteomic approach to assess the milk species involved in cheese production. Selective peptides were detected in milk to use as markers in cheese products. Trypsin-digested milk samples of cow, sheep, goat, and buffalo, analyzed by HPLC-tandem mass spectrometry provided species-specific peptides, some of them recognized by Mascot software (Matrix Science Ltd., Boston, MA) as derived from well-known species specific proteins. A multianalyte multiple reaction monitoring method, built with these specific peptides, was successfully applied to cheeses with different composition, showing high specificity in detection of species involved. Neither aging nor production method seemed to affect the response, demonstrating that chosen peptides well act as species markers for dairy products.

Evolution of species-specific major seminal fluid proteins in placental mammals by gene death and positive selection

The seminal fluid is a complex substance composed of a variety of secreted proteins and has been shown to play an important role in the fertilisation process in mammals and also in Drosophila. Several genes under positive selection have been documented in some rodents and primates. Our study documents this phenomenon in several other mammalian taxa. We study the evolution of genes that encode for 20 proteins that are quantitatively predominant in the seminal fluid of at least one out of seven domestic animal species. We analyse the amino acid composition of these proteins for positive selection and for the presence of pseudogenes. Genes that disappeared through pseudogenisation include KLK2 in cattle, horse and mice. Traces of positive selection are found in seven genes. The identified amino acids are located in regions exposed to the protein surface, suggesting a role in the interaction of gametes, with possible impact on the process of speciation. Moreover, we found no evidence that the predominance of proteins in seminal fluid and their mode of evolution are correlated, and the uncoupled patterns of change suggest that this result is not due solely to lack of statistical power.

In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae.

Recent proteome analyses have reported that intrinsically disordered regions (IDRs) of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs) has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST) and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.

Probing the Biology of Giardia intestinalis Mitosomes Using In Vivo Enzymatic Tagging.

Giardia intestinalis parasites contain mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. An in vivo enzymatic tagging technique based on the Escherichia coli biotin ligase (BirA) has been introduced to G. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import were in vivo tagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific to G. intestinalis and even absent from the related diplomonad parasite Spironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. The in vivo enzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport.

A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS)

Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with 34S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and 34S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (msp/msam) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5–3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations.

Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport.

It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (Kp) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The Kp for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The Kp for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies.

Development of Recombinant Leptospira Antigenes for the Purpose of Creation of Immunoassay Test Kit for Diagnosis of Leptospirosis

Species-specific recombinant proteins of leptospira are genetically engineered by the method of gene manipulation. Their high activity and antigenic specificity are established. On the basis of these proteins an immunoassay test kit for detecting JgG to Leptospira is constructed.

Toll-like receptor signaling in vertebrates: testing the integration of protein, complex, and pathway data in the protein ontology framework.

The Protein Ontology (PRO) provides terms for and supports annotation of species-specific protein complexes in an ontology framework that relates them both to their components and to species-independent families of complexes. Comprehensive curation of experimentally known forms and annotations thereof is expected to expose discrepancies, differences, and gaps in our knowledge. We have annotated the early events of innate immune signaling mediated by Toll-Like Receptor 3 and 4 complexes in human, mouse, and chicken. The resulting ontology and annotation data set has allowed us to identify species-specific gaps in experimental data and possible functional differences between species, and to employ inferred structural and functional relationships to suggest plausible resolutions of these discrepancies and gaps.

GASS: genome structural annotation for Eukaryotes based on species similarity.

BackgroundWith the development of high-throughput sequencing techniques, more and more genomes were sequenced and assembled. However, annotating a genome’s structure rapidly and expressly remains challenging. Current eukaryotic genome annotations require various, abundant supporting data, such as: species-specific and cross-species protein sequences, ESTs, cDNA and RNA-Seq data. Collecting those data and merging their analytical results to achieve a consistent complete annotation is a complex, time and cost consuming task.ResultsIn our study, we proposed a fast and easy-to-use computational tool: GASS (Genome Annotation based on Species Similarity). It annotates a eukaryotic genome based on only the annotations from another similar species. With aligning the exons’ sequences of an annotated similar species to the un-annotated genome, GASS detects the optimal transcript annotations with a shortest-path model. In our study, GASS was used to achieve the rhesus annotations based on the human annotations. The produced annotations were evaluated by comparing them to the two existing rhesus annotation databases (RefSeq and Ensembl) directly and being aligned with three RNA-Seq data of rhesus. The experiment results showed that more than 65% RefSeq exons and splicing junctions were exactly found by GASS. GASS’s sensitivity was higher than RefSeq’s, and was close to Ensembl’s. GASS had higher specificities than Ensembl at gene, transcript, exon and splicing junction levels. We also found the mis-assemblies of rheMac3 genome, which led to the 2 bp shifts in annotating position on exons’ boundary and then the incomplete splicing canonical sites in Refseq annotations. These detections were further supported by various data sources.ConclusionsGASS quickly produces structural genome annotations in sufficient abundance and accuracy. With simple and rapid running of GASS, small labs can create quick views of genome annotations for an un-annotated species, without the necessity to create, collect, analyze and synthesize extra various data sources, or wait several months for the annotations from professional organizations. GASS can be applied to many study occasions, such as the analysis of RNA-Seq datasets from the unannotated species whose genome drafts are available but the annotations are not.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1353-3) contains supplementary material, which is available to authorized users.

Comparative analysis of predicted plastid-targeted proteomes of sequenced higher plant genomes.

Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding plastid biology, preferentially based on model systems is deficient. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids.

Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF-MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.

Structural libraries of protein models for multiple species to understand evolution of the renin-angiotensin system

The details of protein pathways at a structural level provides a bridge between genetics/molecular biology and physiology. The renin-angiotensin system is involved in many physiological pathways with informative structural details in multiple components. Few studies have been performed assessing structural knowledge across the system. This assessment allows use of bioinformatics tools to fill in missing structural voids. In this paper we detail known structures of the renin-angiotensin system and use computational approaches to estimate and model components that do not have their protein structures defined. With the subsequent large library of protein structures, we then created a species specific protein library for human, mouse, rat, bovine, zebrafish, and chicken for the system. The rat structural system allowed for rapid screening of genetic variants from 51 commonly used rat strains, identifying amino acid variants in angiotensinogen, ACE2, and AT1b that are in contact positions with other macromolecules. We believe the structural map will be of value for other researchers to understand their experimental data in the context of an environment for multiple proteins, providing pdb files of proteins for the renin-angiotensin system in six species. With detailed structural descriptions of each protein, it is easier to assess a species for use in translating human diseases with animal models. Additionally, as whole genome sequencing continues to decrease in cost, tools such as molecular modeling will gain use as an initial step in designing efficient hypothesis driven research, addressing potential functional outcomes of genetic variants with precompiled protein libraries aiding in rapid characterizations.

Leptospira species and serovars identified by MALDI-TOF mass spectrometry after database implementation.

BackgroundLeptospirosis, a spirochaetal zoonotic disease of worldwide distribution, endemic in Europe, has been recognized as an important emerging infectious disease, though yet it is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries. Leptospirosis is caused by the infection with any of the more than 230 serovars of pathogenic Leptospira sp. In this study we aimed to implement the MALDI-TOF mass spectrometry (MS) database currently available in our laboratory with Leptospira reference pathogenic (L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchii), intermediate (L. fainei) and saprophytic (L. biflexa) strains of our collection in order to evaluate its possible application to the diagnosis of leptospirosis and to the typing of strains. This was done with the goal of understanding whether this methodology could be used as a tool for the identification of Leptospira strains, not only at species level for diagnostic purposes, but also at serovar level for epidemiological purposes, overcoming the limits of serological and molecular conventional methods. Twenty Leptospira reference strains were analysed by MALDI-TOF MS. Statistical analysis of the protein spectra was performed by ClinProTools software.ResultsThe spectra obtained by the analysis of the reference strains tested were grouped into 6 main classes corresponding to the species analysed, highlighting species-specific protein profiles. Moreover, the statistical analysis of the spectra identified discriminatory peaks to recognize Leptospira strains also at serovar level extending previously published data.ConclusionsIn conclusion, we confirmed that MALDI-TOF MS could be a powerful tool for research and diagnostic in the field of leptospirosis with broad applications ranging from the detection and identification of pathogenic leptospires for diagnostic purposes to the typing of pathogenic and non-pathogenic leptospires for epidemiological purposes in order to enrich our knowledge about the epidemiology of the infection in different areas and generate control strategies.

Rapid and reliable species identification of scallops by MALDI-TOF mass spectrometry

Correct identification of scallop species is of crucial importance to the seafood industry to assess marketability of the product, as well as to prevent unintentional mislabeling and to comply with current legislative guidelines. However, various scallop species are virtually indistinguishable and to date there is no fast, reproducible, and inexpensive technique for species identification of scallops. A new promising tool in the identification of various organisms is the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In this study, we aimed to 1) evaluate the possibility of MALDI-TOF MS as a tool for rapid and reliable species identification of scallops and 2) assess the occurrence of mislabeled scallops sold in Switzerland. We therefore used 16 scallop reference samples, as well as scallops sampled at eight Swiss retail facilities and identified the scallop species both by 16S rRNA PCR and MALDI-TOF MS. For all tested scallop species, MALDI-TOF MS yielded reproducible and unique mass spectra profiles covering a wide molecular mass range of 3000–30,000 Da. Species-specific biomarker protein mass patterns were determined. Species identification of reference scallop samples using MALDI-TOF MS was in accordance with species identification using the established 16S rRNA PCR tool. Identification of the scallop samples purchased in Switzerland also yielded identical results for MALDI-TOF MS and the 16S RNA PCR approach. We were able to show that several of the scallop samples were labeled incorrectly. All mislabeled scallops belonged to the species Placopecten magellanicus, but were marketed as “Jacobsmuschel”, a commercial name reserved for Pecten spp. In this study, we show that MALDI-TOF MS allows for rapid, reliable, and highly accurate species identification of scallops.

Benchmarking of Methods for Genomic Taxonomy

One of the first issues that emerges when a prokaryotic organism of interest is encountered is the question of what it is--that is, which species it is. The 16S rRNA gene formed the basis of the first method for sequence-based taxonomy and has had a tremendous impact on the field of microbiology. Nevertheless, the method has been found to have a number of shortcomings. In the current study, we trained and benchmarked five methods for whole-genome sequence-based prokaryotic species identification on a common data set of complete genomes: (i) SpeciesFinder, which is based on the complete 16S rRNA gene; (ii) Reads2Type that searches for species-specific 50-mers in either the 16S rRNA gene or the gyrB gene (for the Enterobacteraceae family); (iii) the ribosomal multilocus sequence typing (rMLST) method that samples up to 53 ribosomal genes; (iv) TaxonomyFinder, which is based on species-specific functional protein domain profiles; and finally (v) KmerFinder, which examines the number of cooccurring k-mers (substrings of k nucleotides in DNA sequence data). The performances of the methods were subsequently evaluated on three data sets of short sequence reads or draft genomes from public databases. In total, the evaluation sets constituted sequence data from more than 11,000 isolates covering 159 genera and 243 species. Our results indicate that methods that sample only chromosomal, core genes have difficulties in distinguishing closely related species which only recently diverged. The KmerFinder method had the overall highest accuracy and correctly identified from 93% to 97% of the isolates in the evaluations sets.

Comparing fragmentation spectra from two parasitic worm species to discover unique peptides

Mass spectrometry (MS) is a technique used in proteomics to identify proteins. Fragmentation spectra (MS2) are commonly assigned to peptides by database searches to identify proteins. Assignment of identified peptides to a genome can be achieved by the field of proteogenomics. Complete genomes, and therefore derived proteome databases, are available for some well-studied species but not for all other species. Taenia solium and Taenia hydatigena are two closely related tapeworm species with limited and almost complete lack of sequenced genome. The larval stages of both these parasites can infect pigs, while humans can only be infected by T. solium. When investigating the prevalence of T. solium in pigs, one needs a diagnostic test that is able to distinguish between the two Taenia species. Current tests, however, cannot make any distinctions. A possible solution would be identifying species-specific proteins for a possible diagnostic test despite limitations at genome-level. To achieve this, we compare spectra between datasets from each species to determine unique spectra. Full proteogenomics searches are then used as a follow-up for interesting leads.

Effect of Processing on PCR Detection of Animal Species in Meat Products

Although most consumers are sensitive about the origin of the meat they consume, adulteration of meat products is not uncommon. For this reason, the development of reliable methods for animal species identification in meat products is an important research priority for food scientists. Species-specific protein- and DNA- identification methods generally used for this purpose. ELISA and protein electrophoresis are used for protein, PCR is used for species-specific DNA identification. Because DNA is known to be more resistant to processing than protein, PCR methods are generally considered as more sensitive for processed foods. However, processing conditions may also degrade DNA resulting in decreased DNA quality and yield. In this study, the individual and combined effects of heat treatment and low pH on the identification of animal species in meat products by PCR were evaluated. Beef sausage mixtures containing two different amounts of meat from a secondary species (either poultry, pork, or horse) were prepared and were subjected to heat treatment (65°C, 85°C, and 121°C) and pH adjustment (5.2 and 6.2). PCR screening for the four animal species was performed using DNA extracts of these meat samples. The results showed that, the combined effect of high temperature and low pH significantly affects the detection limit of the PCR method. Nevertheless, even low levels of adulteration can still be detected fallowing heat treatment.

The application of proteomic technologies for the analysis of muscle proteins of farm animals used in the meat industry (review)

The review briefly summarizes the data on the development of proteomic technologies that became actively used in studies of the muscular proteins of farm animals used in the meat industry in 2006-2013. It has been noted that the main research trends are connected with the detection of changes in muscle proteins during post-mortem autolysis and the search for species-specific and other protein biomarkers. Particular publications regarding the development of methods based on proteomic technologies for monitoring the state of muscle proteins are considered. According to the analyzed data, we can conclude that the field is promising for the solution of a number of pressing problems in.applied biochemistry.

Identification of phlebotomine sand flies (Diptera: Psychodidae) by matrix-assisted laser desorption/ionization time of flight mass spectrometry

BackgroundPhlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens. To elucidate their role as vectors, proper species identification is crucial. Since traditional morphological determination is based on minute and often dubious characteristics on their head and genitalia, which require certain expertise and may be damaged in the field-collected material, there is a demand for rapid, simple and cost-effective molecular approaches.MethodsSix laboratory-reared colonies of phlebotomine sand flies belonging to five species and four subgenera (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius) were used to evaluate the discriminatory power of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Various storage conditions and treatments, including the homogenization in either distilled water or given concentrations of formic acid, were tested on samples of both sexes.ResultsSpecimens of all five analysed sand fly species produced informative, reproducible and species-specific protein spectra that enabled their conclusive species identification. The method also distinguished between two P. sergenti colonies originating from different geographical localities. Protein profiles within a species were similar for specimens of both sexes. Tested conditions of specimen storage and sample preparation give ground to a standard protocol that is generally applicable on analyzed sand fly specimens.ConclusionsSpecies identification of sand flies by MALDI-TOF MS is feasible and represents a novel promising tool to improve biological and epidemiological studies on these medically important insects.

A simple SDS‐PAGE protein pattern from pitcher secretions as a new tool to distinguish Nepenthes species (Nepenthaceae)

Carnivorous plants have always fascinated scientists because these plants are able to attract, capture, and digest animal prey using their remarkable traps that contain digestive secretions. Nepenthes is one of the largest genera of carnivorous plants, with 120 species described thus far. Despite an outstanding diversity of trap designs, many species are often confused with each other and remain difficult to classify because they resemble pitchers or of the occurrence of interspecific hybrids. Here, we propose a new method to easily distinguish Nepenthes species based on a SDS PAGE protein pattern analysis of their pitcher secretions. Intraspecific comparisons were performed among specimens growing in different environmental conditions to ascertain the robustness of this method. Our results show that, at the juvenile stage and in the absence of prey in the pitcher, an examined species is characterized by a specific and stable profile, whatever the environmental conditions. The method we describe here can be used as a reliable tool to easily distinguish between Nepenthes species and to help with potential identification based on the species-specific protein pattern of their pitcher secretions, which is complementary to the monograph information.