Protein Extraction Protocol Research Articles

Optimization of protein extraction for proteomic analysis of formalin-fixed and paraffin-embedding (FFPE) biopsy specimens from males with breast cancer.

e13119 Background: Male breast cancer corresponds to 1% of breast cancer diagnoses. Given this low incidence, the tumors detected have a worse prognosis and lethality. Formalin fixation and paraffin-embedding (FFPE) methods are the most common to maintain biopsied samples. It is a low-cost procedure and is a valuable resource for the study of pathological molecular mechanisms and research of potential biomarkers and therapeutic target molecules. However, in this technique cross-links are formed compromising protein extraction, resulting in low yield and quality for mass spectrometry analysis. Objective: To present an optimized method of protein extraction from male breast FFPE samples for proteomic analysis. Methods: Four protein extraction protocols were optimized and tested in female breast cancer FFPE samples from Haroldo Juaçaba Hospital/Cancer Institute of Ceará to preserve the male sample. Each protocol was performed using a total of eight 5uM tumor slides. Different deparaffinization processes were tested followed by distinct extraction buffers and variable incubation time. The characteristics of each of the protocols and optimizations are presented in the table. The quantification of the protein profile was carried out in nanodrop 2000. The protocol with the highest yield was used in the male breast cancer sample FFPE. Analysis of the protein profile in polyacrylamide gel (SDS PAGE) was performed. Results: Protocol IV presented the highest yield of protein (16.9 mg/mL) and protocols I, II, and III presented, 0.46 mg/mL and 0.28 mg/ml, and 2.3 mg/mL, respectively. Protocol IV was selected to be used in the male breast cancer sample and the yield was 14.26 mg/mL, similar to the female sample. Protein bands from male and female samples using protocol IV were detected on the SDS-PAGE gel. Conclusions: Protocol IV performed a satisfactory protein yield in breast cancer samples FFPE. [Table: see text]

Evaluation and clinical impact of MALDI Biotyper Mycobacteria Library v6.0 for identification of nontuberculous mycobacteria by MALDI‐TOF mass spectrometry

Major challenges in the identification of non-tuberculous mycobacteria (NTM) by MALDI-TOF MS include protein extraction protocol and updating of the NTM database. The aim of this study was to evaluate MALDI Biotyper Mycobacteria Library v6.0 (Bruker Daltonics GmbH, Bremen, Germany) for identification of clinical NTM isolates and its impact on clinical management. NTM isolates cultivated from clinical samples in 101 patients were identified simultaneously by PCR-reverse hybridization (Hain Lifescience GmbH, Nehren, Germany) as a routinely used reference molecular method and using MALDI Biotyper Microflex LT/SH after protein extraction. Each isolate was applied to eight spots, and mean scores were used in analysis. MALDI-TOF MS obtained correct identification to the species level for 95 (94.06%) NTM isolates. The majority of correctly identified isolates (92/95; 96.84%) were identified with high-confidence score of ≥1.80 and only 3.16% (3/95) with a score of <1.80. Mean value ± SD of RGM NTM isolates (2.127 ± 0.172) was statistically significant higher in comparison to SGM NTM isolates (2.027 ± 0.142) with a p value of 0.007. In comparison to PCR-reverse hybridization, discordant identification results by MALDI-TOF MS were found in six (6/101; 5.94%) NTM isolates for which clinical data were analyzed. We demonstrated a high confidence NTM identifications using Mycobacterium Library v 6.0 on routine clinical isolates. This is the first study that analyzed MALDI-TOF MS identification results of NTM isolates in the context of clinical data, and it showed that MALDI-TOF MS with its updated databases could help clarify the epidemiology, clinical characteristics, and course of infections caused by less frequent NTM species.

Comparison of different protocols for protein extraction from murine ovarian tissue: A preliminary study

Objective: Extraction of proteins from tissues is a key step to study the protein structure and function. There is no set of universal protocols because of the enormous chemical and physical variety of both proteins and the sample sources. Hence, in this study a comparison of different methods for protein extraction from mouse ovarian tissue was carried out. Methods: Intravaginal dose of 20µl of Phosphate buffer saline was given to 7 mice for ten consecutive days followed by extraction of ovaries. For comparison, seven different protein extraction protocols from Protocol 1 to Protocol 7 were used. The supernatant was first quantified with Bradford assay to determine the total protein yield followed by SDS-PAGE analysis. Results: Highest protein yield of 1.13 mg/ml was attained after supplementing 1% v/v protease inhibitor cocktail in PBS. In total, 13 protein bands were detected by the extraction protocol 2 and 11 protein bands by the extraction protocol 1. 11 protein bands were detected in the range 180 kDa to 22 kDa following Protocol 3 and 4. Extraction with lysis buffer 1 resulted in 7 visible protein bands of molecular weight 182 kDa to 35 kDa range. Only 6 protein bands were observed in the range 182 kDa - 35 kDa following lysis buffer 2 method. For lysis buffer 3, ten visible protein bands were detected in range of 195 kDa - 13 kDa. Conclusion: Based on the protein extraction efficiency of Protocol 1, optimal combination of Phosphate buffer saline (pH 7.2) and protease inhibitor cocktail followed by hand-homogenization is suggested for protein extraction from mouse tissues.

MALDI-TOF MS as a tick identification tool in a tertiary hospital in Spain

In Spain, as in other countries, the spectrum of tick-borne diseases and their number have increased in recent years. The tick identification, at species level, can be challenging outside research centers although this information is very usufull for decisions making. The performance of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in tick identification of specimens collected from patients have been seldomly reported. The aim of the present study was to desing a protein-extraction protocol and build a tick-legs reference spectra. This protocol was then validated using specimens from both patients and non-patient sources. Nine species of ticks that usually bites humans in Spain were included: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis punctata, Hyalomma lusitanicum, Hyalomma marginatum, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus pusillus and Rhipicephalus sanguineus sensu lato. Other less-frequent biting species were also included: Haemaphysalis inermis, Haemaphysalis concinna, Hyalomma scupense, Ixodes frontalis, Ixodes hexagonus, and Argas sp. specimens were identified by PCR and sequencing of a fragment of the 16S rRNA gene of ticks. In the tests performed with non-patient collected specimens, a 100% correlation was observed between molecular methods and MS, while in the tests performed with ticks collected from patients a 92.59% correlation was observed. Misidentification was observed only in two of I. ricinus nymphs (identified as Ctenocephalides felis). Therefore, mass- spectrometry can be confidently used as a tick identification tool in a hospital setting for the rapid identification of tick vectors.

Preparation and nutritional characterisation of protein concentrate prepared from foxtail millet (Setaria italica).

Plant-based protein sources as a sustainable alternative to animal sources are highly relevant for food and dietary supplements industries. Plant proteins are becoming popular as an eco-friendly source for meeting global protein requirements due to their importance in nutrition, management of metabolic diseases, biological activities, functionality in processed food products and their low carbon footprints. We applied biochemical protein extraction protocol and prepared protein concentrate from an underutilised cereal, foxtail millet, with plausible applications in foods and supplements. Herein efforts were utilised to obtain foxtail millet protein (FMP) concentrate by means of standardisation of processes of extraction cum isolation. The conditions including flour to solvent ratio, extraction-precipitation pH, dissolution time, etc. were optimised to significantly improve protein yield and recovery. The FMP concentrate prepared was also analysed for nutritional composition, bioactive compounds, amino acid content and digestion properties in comparison to packaged brown rice protein concentrate. The protein concentrate prepared was found to have high digestibility, rich in essential amino acids with good phenolic and flavonoid content, thereby making it a potential sensory and antioxidant additive for food/pharmaceutical applications.

Unpacking the Proteome and Metaproteome of the Black Soldier Fly Larvae: Efficacy and Complementarity of Multiple Protein Extraction Protocols.

The larvae of the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), have demonstrated the ability to efficiently bioconvert organic waste into a sustainable source of food and feed, but fundamental biology remains to be discovered to exploit their full biodegradative potential. Herein, LC-MS/MS was used to assess the efficiency of eight differing extraction protocols to build foundational knowledge regarding the proteome landscape of both the BSF larvae body and gut. Each protocol yielded complementary information to improve BSF proteome coverage. Protocol 8 (liquid nitrogen, defatting, and urea/thiourea/chaps) was better than all other protocols for the protein extraction from larvae gut samples, and the exclusion of defatting steps yielded the highest number of proteins for the larval body samples. Protocol-specific functional annotation using protein level information has shown that the selection of extraction buffer can affect protein detection and their associated functional classes within the measured BSF larval gut proteome. A targeted LC-MRM-MS experiment was performed on the selected enzyme subclasses to assess the influence of protocol composition using peptide abundance measurements. Metaproteome analysis of the BSF larvae gut has uncovered the prevalence of two bacterial phyla: actinobacteria and proteobacteria. We envisage that using complementary extraction protocols and investigating the proteome from the BSF body and gut separately will expand the fundamental knowledge of the BSF proteome and thereby provide translational opportunities for future research to enhance their efficiency for waste degradation and contribution to the circular economy.

Differential analysis of the dentin soluble proteomic

ObjectivesTo perform a differential analysis of the dentin soluble proteomic and assess the effects of tissue health state and protocol for protein extraction. We hypothesized the dentin soluble proteomic varies according to the tissue physiopathological state (intact vs. caries-affected) and protocol used to extract its proteins. MethodsDentin from freshly extracted non-carious and carious teeth were randomly assigned for protein extraction using either guanidine-HCl/ethylenediaminetetraacetic acid (EDTA) or acetic acid. Protein extracts from intact and caries-affected dentin were processed and digested with trypsin for shotgun label-free proteomic analysis (nLC-ESI-MS/MS). Peptides identification was performed on a nanoACQUITY UPLC-Xevo Q-Tof MS system. Peptides identified with scores of confidence greater than 95% were included in the quantitative statistical analysis embedded in the PLGS software. Differences between experimental conditions were calculated using Student test-t with significance pre-set at α=0.05. ResultsA total of 158 human proteins were identified. Approximately one-sixth of proteins (24/158) were present in at least two different extracts. Conversely, the greatest number of proteins (134/158) was identified uniquely in only one of the extracts. Overall, a larger number of soluble proteins was retrieved from caries-affected than intact dentin (86/158). Likewise, a greater number of proteins was extracted by the guanidine-HCl/EDTA (106/158) in comparison to acetic acid protocol. Several proteins detected in dentin extracts, mainly those from caries-affected teeth, are biological and/or metabolically involved with tissue turnover/remodeling. ConclusionThe identity/abundance of soluble proteins retrieved from and remained in dentin noticeably depend on this tissue physiopathological state and protocol used to remove its minerals. Clinical significanceThe present findings brought new insight into the proteomic phenotype of human dentin and may provide targets for the development of novel caries disease-prevention therapies.

Getting Ready for Large-Scale Proteomics in Crop Plants.

Plants are an indispensable cornerstone of sustainable global food supply. While immense progress has been made in decoding the genomes of crops in recent decades, the composition of their proteomes, the entirety of all expressed proteins of a species, is virtually unknown. In contrast to the model plant Arabidopsis thaliana, proteomic analyses of crop plants have often been hindered by the presence of extreme concentrations of secondary metabolites such as pigments, phenolic compounds, lipids, carbohydrates or terpenes. As a consequence, crop proteomic experiments have, thus far, required individually optimized protein extraction protocols to obtain samples of acceptable quality for downstream analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). In this article, we present a universal protein extraction protocol originally developed for gel-based experiments and combined it with an automated single-pot solid-phase-enhanced sample preparation (SP3) protocol on a liquid handling robot to prepare high-quality samples for proteomic analysis of crop plants. We also report an automated offline peptide separation protocol and optimized micro-LC-MS/MS conditions that enables the identification and quantification of ~10,000 proteins from plant tissue within 6 h of instrument time. We illustrate the utility of the workflow by analyzing the proteomes of mature tomato fruits to an unprecedented depth. The data demonstrate the robustness of the approach which we propose for use in upcoming large-scale projects that aim to map crop tissue proteomes.

Untargeted Proteomics and Metabolomics Analysis of Plant Organ Development.

Our understanding of major developmental transitions in plants and animals has been transformed by the emergence of omics technologies. The majority of leaf growth research has been conducted at the transcriptional level. Although historically understudied, alterations at the protein and metabolite levels have begun to gain traction in recent years. Here, we present a protocol for metabolite and protein extraction followed by untargeted metabolomics and proteomics analysis of the growing leaves.

The Hidden Secrets of the Dental Calculus: Calibration of a Mass Spectrometry Protocol for Dental Calculus Protein Analysis.

Dental calculus is a solid deposit that forms and accumulates on the tooth surface, entrapping oral microorganisms, biomolecules, and other micro-debris found in the oral cavity. A mass spectrometry analysis of its protein content opens a vista into the subject's diet, oral flora, and even some aspects of health, thus providing new insight and expanding our knowledge of archaic cultures. Multiple experimental protocols have been proposed for the optimal extraction of proteins from dental calculus. Herein, we compared various experimental conditions in order to calibrate and validate a protocol for protein extraction. Our results show that a high concentration of acetic acid followed by mechanical crushing and sonication provided the highest protein yield, while acetone precipitation enabled the identification of more distinct proteins. We validated this protocol using archeological samples, identifying human and microbial proteins in specimens from the eighth and seventeenth centuries (approximately 250-1300 years ago). These findings demonstrate that the developed protocol is useful for studying excavated archaeological samples and that it might be utilized to explore the biohistory, dietary habits, and microbiome of archaic populations.

Rapeseed Cake Valorization into Bioplastics Based on Protein Amyloid Fibrils

AbstractBiodegradable polymers obtained from renewable resources are urgently needed to help mitigating the environmental burden of plastic pollution. Herein, a green process is developed to obtain protein‐based bioplastics through valorization of rapeseed cake. First, the effects of different protein extraction protocols are investigated, including water extraction at pH values from 1 to 12 and salt extraction with isoelectric point precipitation (IP), on proteins profile and sample composition. It is demonstrated that alkaline extraction at pH 12 results in the highest extraction yields of proteins (≈70%) and total solutes (≈50%). The IP results in lower yields but in highest protein purity of ≈80%. Alkaline extraction at pH 10.5 results in the extraction of 36% total solutes and ≈50% of proteins, with a protein profile similar to that obtained by IP, rich in α and β polypeptides chains of cruciferin. AFM analysis shows that samples containing cruciferin allow self‐assembly into mature amyloid fibrils upon incubation. Rapeseed amyloids are blended with polyvinyl alcohol and glycerol to develop bioplastics films that are characterized by decreased water absorption, higher water contact angle, and elongation at break &gt;600%, more than double compared to films obtained from native monomers.

Identification of transient receptor potential melastatin 3 proteotypic peptides employing an efficient membrane protein extraction method for natural killer cells.

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer’s recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150–200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.

Comparison of protein extraction protocols and allergen mapping from black soldier fly Hermetia illucens

Exploration of important insect proteins — including allergens — and proteomes can be limited by protein extraction buffer selection and the complexity of the proteome. Herein, LC-MS/MS-based proteomics experiments were used to assess the protein extraction efficiencies for a suite of extraction buffers and the effect of ingredient processing on proteome and allergen detection. Discovery proteomics revealed that SDS-based buffer yields the maximum number of protein groups from three types of BSF samples. Bioinformatic analysis revealed that buffer composition and ingredient processing could influence allergen detection. Upon applying multi-level filtering criteria, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. A targeted LC-MRM-MS assay was developed for the pan-allergen tropomyosin and used to assess the influence of buffer composition and ingredient processing using peptide abundance measurements. SignificanceWe demonstrated that the selection of protein extraction buffer and the processing method could influence protein yield and cross-reactive allergen detection from processed and un-processed black soldier fly (BSF) samples. In total, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. An LC-MRM-MS assay was developed for tropomyosin, indicating the importance of buffer selection and processing conditions to reduce BSF samples' allergenicity.

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1.

Patients who suffer from birch pollinosis often develop adverse reactions to the consumption of fresh apples due to the structural similarity of the allergens Bet v 1 and Mal d 1 from birch and apples, respectively. A different allergenic potential for Mal d 1 isoallergens is postulated, but approaches to quantify the Mal d 1 isoallergen-specific are missing. Therefore, a bottom-up proteomics approach was developed to quantify Mal d 1 by stable isotope dilution and microHPLC-QTOF analyses. Marker peptides for individual isoallergens (Mal d 1.01-Mal d 1.03 and Mal d 1.06), combinations thereof (Mal d 1.01 + 1.02, Mal d 1.02 + 1.06, and Mal d 1.04 + 1.05), and two global marker peptides, comprising Mal d 1.01 + 1.02 + 1.04 + 1.05 and Mal d 1.03 + 1.06 + 1.07 + 1.08 + 1.09, were identified. By the use of an extraction standard (r-Mal d 1_mut), an optimized protocol for extraction and tryptic digestion of apple proteins was developed, and the variety-specific extraction efficiency was monitored for the flesh and peel of apples. The Mal d 1 contents in flesh and peel of five commercial apple breeds and four apple varieties from orchard meadows were quantified isoallergen-specific.

Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure.

The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome studies is key in clinical translation of the findings. Thus, we aimed to optimize a protocol for aortic valve homogenization and protein extraction and to develop a standard operating procedure (SOP), which researchers can use to maximize protein yield while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenization with a Potter apparatus with a more advanced bead-beating system. Once we confirmed the superiority of the latter, we further optimized it by testing the effect of beads size, the number of homogenization cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenization cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2 mL O-ring cryotubes. As a proof of concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterized, resulting in the quantification of proteins across six orders of magnitude and uncovering some putative proteins dysregulated by sex.

Faster and accurate identification of clinically important Trichosporon using MALDI TOF MS

PurposeTrichosporon species are emerging human pathogens, accounting for the second most common cause of non-candidal mycosis. Rapid and reliable identification of these agents allows a better understanding of their epidemiology and therapeutic management. The Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technique has the potential to be precise, fast and cost-effective. However, the precision of identification totally depends upon the type of protein extraction method used and embedded database in the system. Our objectives were to standardize the protein extraction technique and expand the present Bruker database by creating an in-house database and validating it with diverse clinical Trichosporon species of Indian origin. MethodsTwo different protein extraction protocols (on-plate and off-plate) were evaluated. The off-plate protocol was finalized for the identification. MALDI TOF MS with the existing Bruker database was evaluated for its ability to identify a total of 79 intergenic spacer 1 (IGS1) gene sequence confirmed clinical isolates of 5 different Trichosporon species. ResultsAs outcome, off plate protocol yielded higher accuracy (73% on the species level and 95% on the genus level) than on-plate (25% on the genus level) in terms of log scores. The existing database for Trichosporon species was enriched with 28 sequence confirmed isolates, which improved accuracy from 73% to 100% and were identified up to species level with a log score >2.3. ConclusionsUsed with standardized protein-extraction protocol along with an expanded database, MALDI-TOF MS could be a rapid and reliable approach to identify clinical Trichosporon species routinely in the laboratory.

MALDI-TOF MS application for identification of filamentous fungi

Introduction. MALDI-TOF identification method is widely used in microbiology due to its accuracy and rapid results achievement. However, applying this method to mold fungi faces some difficulties and is not always effective. Purpose of the study. The aim of the study was to evaluate the profits of using the long cultivation and protein extraction protocol in routine identification of mold fungi isolates from environment. Materials and methods. The analysis of molds museum collection from Centre for Strategic Planning of FMBA of Russia was performed by MALDI-TOF mass-spectrometry Biotyper (Bruker Daltonics) with cultivation in liquid media and long optimized protein extraction protocol with acetonitrile and formic acid. Results. One hundred thirty seven isolates were analyzed. Quality spectra were achieved for 71.5% of samples. Identification with MBT Filamentous Fungi Library database with the high confidence score (&gt; 1.7) was achieved for 55% of isolates (26% with score &gt;2). Samples analyzed included members of nineteen families and 27 genera. 16% of samples were not identified despite producing high-quality spectra. Limitations. When studying the possibility of using the time-of-flight mass spectrometry method to identify mold fungi, a sample of 137 isolates of mold fungi from the environment was analyzed, which is a sufficient reference sample. The analyzed samples included representatives of 19 families and 27 genera, which makes it possible to apply the findings to at least these representatives of micellar fungi. In this study 22 samples with good quality spectra, were not identified with MBT Filamentous Fungi Library database. In the future studies, these samples, along with other samples like that, will be identified by genetic molecular methods and added to the new home-made database for filamentous fungi MALDI-TOF identification. Conclusion. Effective identification of filamentous fungi by mass-spectrometry methods requires pure culture achieved from liquid media, long optimized protocol of protein extraction and building an in-house database of spectra not presented in Bruker database.

Systematic evaluation and optimization of protein extraction parameters in diagnostic FFPE specimens

ObjectivesFormalin-fixed paraffin-embedded (FFPE) tissue is the standard material for diagnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction protocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of functionally relevant proteins.MethodsUsing the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was measured by protein concentration and western blot analysis of cellular compartment markers as well as liquid chromatography-coupled mass spectrometry (LC–MS).ResultsCommercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval.ConclusionsPreanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.

Investigation of The Efficacy of Three Different Protein Extraction Protocols and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Nontuberculous Mycobacteria

There are more than 160 defined nontuberculous mycobacteria (NTM) species within Mycobacterium genus. In recent years, the number of NTM species associated with human infections and the infections caused by them have been reported at increasing rates. The identification of these species by phenotypic methods is difficult, laborious, and unlikely to obtain reliable results. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) MALDI-TOF MS has proven to be a good method for the identification of bacteria and yeasts in routine laboratory practices. However, Mycobacterium species differ from other bacteria by their cell wall structures, less ribosomal protein content, and slower growth rates. A standardized and efficient protein extraction protocol for MALDI-TOF MS analysis of mycobacteria is essential. The aim of our study was to investigate the efficacy of different protein extraction protocols and the MALDI-TOF MS method in the diagnosis of NTM species. A total of 73 NTM isolates, grown in both solid and liquid media and previously identified with line probe assay, were evaluated with MALDI-TOF MS (Bruker Daltonics GmbH and Co. KG, Germany). Stock isolates were homogenized and decontaminated by N-Acetyl L-cysteine (NALC)/Sodium hydroxide (NaOH) method. For solid media, isolates were inoculated on Löwenstein-Jensen medium and incubated at 35˚C in a normal atmosphere. For liquid media culture, BD BACTEC MGIT 960 automated system (Becton, Dickinson, Sparks, MD, USA) was performed according to the manufacturer's instructions. For the identification of all isolates by MALDI-TOF MS, the manufacturer's recommended protein extraction protocol (Protocol 1) was compared with the two other protocols, using a simplified extraction procedure (Protocol 2), and freezing temperature (Protocol 3). In the liquid media analysis, the rates of the isolates identified by MALDI-TOF MS (score≥ 2.0) for Protocol 1, 2, and 3 were found as 84.93% (n= 62), 63.01% (n= 46), and 43.83% (n= 32), respectively. In the solid media analysis, the rates of the isolates with an identification score of ≥ 2.0 for the protocols with the same order were determined as 87.67% (n= 64), 52.05% (n= 38), and 31.50% (n= 23), respectively. Isolates grown in both solid and liquid media were identified in the same species level in all three protocols, regardless of the identification values and misidentification was not presented. When the reliable identification score was evaluated as ≥ 2.0 in our study, the manufacturer's recommended MYCOEX IVD procedure was found to be the most effective method for the isolates grown in both liquid and solid media. In conclusion, MALDI-TOF MS has the potential to be a reliable, easy-to-use and fast method that can be used in routine practice for the identification of NTM species with its standardized protein extraction protocols.

Classification of Environmental Strains from Order to Genus Levels Using Lipid and Protein MALDI-ToF Fingerprintings and Chemotaxonomic Network Analysis.

During the last two decades, MALDI-ToF mass spectrometry has become an efficient and widely-used tool for identifying clinical isolates. However, its use for classification and identification of environmental microorganisms remains limited by the lack of reference spectra in current databases. In addition, the interpretation of the classical dendrogram-based data representation is more difficult when the quantity of taxa or chemotaxa is larger, which implies problems of reproducibility between users. Here, we propose a workflow including a concurrent standardized protein and lipid extraction protocol as well as an analysis methodology using the reliable spectra comparison algorithm available in MetGem software. We first validated our method by comparing protein fingerprints of highly pathogenic bacteria from the Robert Koch Institute (RKI) open database and then implemented protein fingerprints of environmental isolates from French Guiana. We then applied our workflow for the classification of a set of protein and lipid fingerprints from environmental microorganisms and compared our results to classical genetic identifications using 16S and ITS region sequencing for bacteria and fungi, respectively. We demonstrated that our protocol allowed general classification at the order and genus level for bacteria whereas only the Botryosphaeriales order can be finely classified for fungi.