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1.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205105

RESUMO

The Late Neolithic palafitte site, Ustie na Drim, in the northern part of Lake Ohrid (North Macedonia), excavated in 1962, offered ceramic fragments of large, flat, elongated pans. These artifacts could be dated by relative chronology to roughly around 5200-5000 BC. According to their shape and technological traits, the ceramic pans were probably used for baking. The attached materials on the surface of studied pan fragments were sampled for consequent chemical and microscopical analyses (i.e., analyses of starch, phytoliths, and microscopic animal remains). An immunological method revealed the presence of pork proteins in samples. The presence of organic residues of animal origin was, moreover, confirmed by the detection of cholesterol using gas chromatography coupled to mass spectrometry. Analysis of detected microscopic botanical objects revealed starch grains of several plants (i.e., oak, cattail, and grasses). An interesting find was the hair of a beetle larva, which could be interpreted contextually as the khapra beetle, a pest of grain and flour. Based on our data, we suppose that the ceramic pans from Ustie na Drim were used for the preparation of meals containing meat from common livestock in combination with cereals and wild plants.


Assuntos
Cerâmica/análise , Alimentos/história , Extratos Vegetais/análise , Proteínas/análise , Animais , Arqueologia , Cerâmica/história , Culinária/história , Cromatografia Gasosa-Espectrometria de Massas , História Antiga , Extratos Vegetais/história , Proteínas/história , República da Macedônia do Norte , Suínos
2.
Toxins (Basel) ; 13(6)2021 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071038

RESUMO

The specificity and potency of venom components give them a unique advantage in developing various pharmaceutical drugs. Though venom is a cocktail of proteins, rarely are the synergy and association between various venom components studied. Understanding the relationship between various components of venom is critical in medical research. Using meta-analysis, we observed underlying patterns and associations in the appearance of the toxin families. For Crotalus, Dis has the most associations with the following toxins: PDE; BPP; CRL; CRiSP; LAAO; SVMP P-I and LAAO; SVMP P-III and LAAO. In Sistrurus venom, CTL and NGF have the most associations. These associations can predict the presence of proteins in novel venom and understand synergies between venom components for enhanced bioactivity. Using this approach, the need to revisit the classification of proteins as major components or minor components is highlighted. The revised classification of venom components is based on ubiquity, bioactivity, the number of associations, and synergies. The revised classification can be expected to trigger increased research on venom components, such as NGF, which have high biomedical significance. Using hierarchical clustering, we observed that the genera's venom compositions were similar, based on functional characteristics rather than phylogenetic relationships.


Assuntos
Venenos de Crotalídeos/análise , Proteínas/análise , Animais , Crotalus/classificação , Crotalus/genética , Filogenia , Toxinas Biológicas/análise
3.
J Chromatogr A ; 1651: 462320, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34144399

RESUMO

This concept article reports a practical solution to improve the linearity of effluent pH response as observed in pH gradient cation exchange chromatography (CEX). When performing pH gradient CEX, it is not easy to develop buffer systems that will universally provide pH response proportional with the mobile phase (buffer) composition. It is an especially challenging pursuit when exploring MS compatible buffers (e.g. ammonium-acetate, ammonium-carbonate). In addition to "non-proportional" behavior from the mobile phase composition, the chromatographic column itself will sometimes impose an unpredictable impact on the effluent pH. Here, we propose a simple approach based on the on-line measurement of effluent pH response, conversion of pH to mobile phase volume fraction (φ) and then generation of the inverse response function in the time domain. In the end, when setting the inverse function as the gradient program instead of a linear gradient, an improved - ideally linear - pH response can be produced. A simple Excel tool was developed to assist analysts with this correction procedure, and it has been made available by download for public use.


Assuntos
Cromatografia por Troca Iônica/métodos , Proteínas/análise , Força Próton-Motriz , Acetatos/química , Anticorpos Monoclonais/análise , Cátions/química , Concentração de Íons de Hidrogênio
4.
J Chromatogr A ; 1651: 462310, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34166860

RESUMO

This study reports on the assessment of the separation performance of hydrophobic interaction chromatography for intact protein analysis using non-porous butyl polymethacrylate phases. The maximum peak capacity in inverse gradient mode was reached at a volumetric flow rate which was significantly (10-20 times) higher than the flow rate yielding the minimum plate height in isocratic mode, as the gradient volume dominates the peak-capacity generation. The flow rate yielding the maximum peak capacity increased with decreasing gradient volume, i.e., steeper gradients, and also depends on the magnitude of the mass-transfer contribution to peak dispersion (affected by particle size and molecular diffusion coefficient of proteins) at these high flow rates. The maximum peak capacity using a 100 mm long column packed with 4 µm particles for steep 7.5 min gradients was determined to be 60. Increasing the column length by coupling columns leads to better gradient performance than increasing the gradient duration for gradients of 60 min and longer. Using a coupled column system (2 × 100 mm long columns packed with 4 µm particles), the maximum peak capacity was determined to be 105, which was 33% higher compared to that of a single column while applying a similar gradient volume. Decreasing the particle size to 2.3 µm leads to higher peak capacities even though the column was operated at lower volumetric flow rate. The maximum peak capacity obtained with the 2.3 µm column was 128% higher than was obtained with the coupled column. Even at suboptimal conditions, the 2.3 µm column yields a higher peak capacity (14%) than when using two coupled columns packed with 4 µm at optimal conditions (gradient time of 120 min and a flow rate of 0.5 mL/min).


Assuntos
Cromatografia/métodos , Interações Hidrofóbicas e Hidrofílicas , Ácidos Polimetacrílicos/química , Proteínas/análise , Animais , Bovinos , Galinhas , Tamanho da Partícula , Ribonuclease Pancreático/metabolismo , Temperatura
5.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068759

RESUMO

Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable 'turn-on' fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules.


Assuntos
Benzotiazóis/química , DNA/análise , Proteínas/análise , Quinolinas/química , DNA/química , Fluorescência , Íons , Sondas Moleculares/química
6.
J Vis Exp ; (170)2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33970141

RESUMO

Förster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas/análise , Animais , Sobrevivência Celular , Microscopia , Ratos
8.
Nat Commun ; 12(1): 2999, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016966

RESUMO

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.


Assuntos
Tecido Adiposo Branco/metabolismo , Proteínas de Membrana/metabolismo , Estado Pré-Diabético/metabolismo , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo Branco/citologia , Adolescente , Adulto , Idoso , Animais , Células CHO , Estudos de Coortes , Cricetulus , Estudos Transversais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevenção & controle , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Insulina/metabolismo , Resistência à Insulina , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Células NIH 3T3 , Estado Pré-Diabético/sangue , Estado Pré-Diabético/tratamento farmacológico , Estado Pré-Diabético/etiologia , Cultura Primária de Células , Proteínas/análise , Receptores Acoplados a Proteínas G/sangue , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Adulto Jovem
9.
J Chromatogr A ; 1647: 462167, 2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-33962076

RESUMO

Simultaneously reducing the bed-height and increasing the area of cross-section, while keeping the bed-volume the same, would substantially reduce the pressure drop across a process chromatography column. This would minimize problems such as resin compaction and non-uniformity in column packing, which are commonly faced when using soft chromatographic media. However, the increase in macroscale convective dispersion due to the increase in column diameter, and the resultant loss in resolution would far outweigh any potential benefit. Cuboid-packed bed devices have lower macroscale convective dispersion compared to their equivalent cylindrical columns. In this paper, we discuss how and why a flat cuboid chromatography device having a short bed-height gives better protein separation, at a significantly lower pressure drop, than a taller column having the same bed-volume. First, we explored this option based on computational fluid dynamic (CFD) simulations. Depending on the flow rate, the pressure drop across the flat cuboid device was lower than that in the tall column by a factor of 6.35 to 6.4 (i.e. less than 1/6th the pressure). The CFD results also confirmed that the macroscale convective dispersion within the flat cuboid device was significantly lower. Head-to-head separation experiments using a 1 mL flat cuboid device having a bed-height of 10 mm, and a 1 mL tall column having a bed-height of 25.8 mm, both packed with the same chromatographic media, were carried out. The number of theoretical plates per unit bed-height was on an average, around 2.5 time times greater with the flat cuboid device, while the total number of theoretical plates in the two devices were comparable. At any given superficial velocity, the height equivalent of a theoretical plate in the tall column was on an average, higher by a factor 2.5. Binary protein separation experiments showed that at any given flow rate, the resolution obtained using the flat cuboid device was significantly higher than that obtained with the tall column. This work opens up the possibility of designing and developing short bed-height chromatography devices for carrying out high-resolution biopharmaceutical purifications, at very low pressures.


Assuntos
Cromatografia/instrumentação , Cromatografia/métodos , Proteínas/isolamento & purificação , Simulação por Computador , Desenho de Equipamento , Proteínas/análise
10.
J Chromatogr A ; 1649: 462234, 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34038775

RESUMO

Today proteins are possibly the most important class of substances. Yet new tasks for proteins are still often solved by trial-and-error approaches. However, in some areas these euphemistically called "screening approaches" are not suitable. E.g. stability tests just take too long and therefore require a more strategic, target-orientated concept. This concept is available by grouping proteins according to their physicochemical properties and then pulling out the right drawer for new tasks. These properties include size, then charge and hydrophobicity as well as their patchinesses, and the degree of order. In addition, solubility, the content of (free) enthalpy, aromatic-amino-acid- and α/ß-frequency as well as helix capping, and corresponding patchiness, the number of specific motifs and domains as well as the typical concentration range can be helpful to discriminate between different groups of proteins. Analyzing correlations will reduce the necessary amount of parameters and additional ones, which may be still undiscovered at the present time, can be identified looking at protein subgroups with similar physicochemical properties which still behave heterogeneously. Step-by-step the methodology will be improved. Possibly protein stability will be the driver of this process, but all other areas such as production, purification and analytics including sample pre-treatment and the choice of appropriate separation conditions for e.g. chromatography and electrophoresis will profit from a rational strategy.


Assuntos
Proteínas/análise , Aminoácidos/análise , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/métodos , Proteínas/química , Solubilidade , Termodinâmica
11.
Methods Mol Biol ; 2228: 1-20, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950479

RESUMO

Mass spectrometry is frequently used in quantitative proteomics to detect differentially regulated proteins. A very important but unfortunately oftentimes neglected part in detecting differential proteins is the statistical analysis. Data from proteomics experiments are usually high-dimensional and hence require profound statistical methods. It is especially important to already correctly design a proteomic experiment before it is conducted in the laboratory. Only this can ensure that the statistical analysis is capable of detecting truly differential proteins afterward. This chapter thus covers aspects of both statistical planning as well as the actual analysis of quantitative proteomic experiments.


Assuntos
Espectrometria de Massas/estatística & dados numéricos , Proteínas/análise , Proteoma , Proteômica/estatística & dados numéricos , Projetos de Pesquisa/estatística & dados numéricos , Animais , Interpretação Estatística de Dados , Humanos , Modelos Estatísticos
12.
Methods Mol Biol ; 2228: 21-28, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950480

RESUMO

Although a lot of new methods for protein concentration determination have been developed and established the last years, the amino acid analysis has still this relevance within proteomics for multiple reasons especially in the quantitative protein analysis. Amino acid analysis enables indirectly both the protein and peptide concentration determination which are essential for using the same amounts for comparative quantitative experiments. Moreover, the quantity and quality of synthetic peptides can be verified. The method itself is robust in comparison with colorimetric assays, especially when detergents or chaotropes are present in the sample buffer. Furthermore, it is highly sensitive. Nevertheless, amino acid analysis needs a certain experience to be set up and is time-consuming compared to other protein concentration determination techniques.


Assuntos
Aminoácidos/análise , Proteínas/análise , Proteoma , Proteômica , Animais , Humanos , Proteólise , Projetos de Pesquisa
13.
Methods Mol Biol ; 2228: 29-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950481

RESUMO

For the quantification of certain proteins of interest within a complex sample, Western blot analysis is the most widely used method. It enables detection of a target protein based on the use of specific antibodies. However, the whole procedure is often very time-consuming. Nevertheless, with the development of fast blotting systems and further development of immunostaining methods, a reduction of the processing time can be achieved. Major challenges for the reliable protein quantification by Western blotting are adequate data normalization and stable protein detection. Usually, normalization of the target protein signal is performed based on housekeeping proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, ß-actin) with the assumption that those proteins are expressed constitutively at the same level across experiments. However, several studies have already shown that this is not always the case making this approach suboptimal. Another strategy uses total protein normalization where the abundance of the target protein is related to the total protein amount in each lane. This approach is independent of a single loading control, and precision of quantification and reliability is increased. For Western blotting several detection methods are available, e.g., colorimetric, chemiluminescent, radioactive, fluorescent detection. Conventional colorimetric staining tends to suffer from low sensitivity, limited dynamic range, and low reproducibility. Chemiluminescence-based methods are straightforward, but the detected signal does not linearly correlate to protein abundance (from protein amounts >5µg) and have a relatively narrow dynamic range. Radioactivity is harmful to health. To overcome these limitations, stain-free methods were developed allowing the combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer to the membrane. Here, we present a rapid Western blot protocol, which combines fast blotting using the iBlot system and fast immunostaining utilizing ReadyTector® all-in-one solution with the Smart Protein Layers (SPL) approach.


Assuntos
Western Blotting , Proteínas/análise , Proteoma , Proteômica , Animais , Western Blotting/normas , Calibragem , Humanos , Proteômica/normas , Padrões de Referência , Projetos de Pesquisa , Fatores de Tempo , Fluxo de Trabalho
14.
Methods Mol Biol ; 2228: 41-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950482

RESUMO

Two-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using two-dimensional gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods, (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of two-dimensional gels is performed.


Assuntos
Eletroforese em Gel Bidimensional , Proteínas/análise , Proteoma , Proteômica , Animais , Corantes Fluorescentes , Humanos , Medições Luminescentes , Projetos de Pesquisa , Coloração e Rotulagem
15.
Methods Mol Biol ; 2228: 53-62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950483

RESUMO

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH-gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures such as Coomassie, silver staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.


Assuntos
Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Proteínas/análise , Proteoma , Proteômica , Animais , Corantes Fluorescentes , Humanos , Concentração de Íons de Hidrogênio , Medições Luminescentes , Projetos de Pesquisa , Coloração e Rotulagem
16.
Methods Mol Biol ; 2228: 63-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950484

RESUMO

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. For its use in proteomics, two important additional features must be considered, compatibility with mass spectrometry and quantitative response. Both features are discussed in this chapter, and optimized silver staining protocols are proposed.


Assuntos
Eletroforese em Gel Bidimensional , Proteínas/análise , Proteoma , Proteômica , Coloração pela Prata , Animais , Humanos , Projetos de Pesquisa
17.
Methods Mol Biol ; 2228: 77-84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950485

RESUMO

Classical 2D-PAGE allows comparison and quantitation of proteomes by visualization of protein patterns using gel stains and comparative image analysis. The introduction of fluorescent reagents for protein labeling (difference in-gel electrophoresis or DIGE) has brought substantial improvement in this field. It provides multiplexing of up to three samples in one gel, higher sensitivity compared to normal protein staining methods, and a higher linear range for quantitation. This article gives detailed protocols for 2D-DIGE, including both minimal and saturation labeling.


Assuntos
Eletroforese em Gel de Poliacrilamida , Proteínas/análise , Proteoma , Proteômica , Eletroforese em Gel Diferencial Bidimensional , Animais , Corantes Fluorescentes , Humanos , Medições Luminescentes , Projetos de Pesquisa , Coloração e Rotulagem
18.
Methods Mol Biol ; 2228: 117-131, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950487

RESUMO

Relative or comparative proteomics provides valuable insights about the altered protein abundances across different biological samples in a single (labeled) or series (label-free) of LC-MS measurement(s). Chemical labeling of peptides using isobaric mass tags for identification and quantification of different proteomes simultaneously has become a routine in the so-called discovery proteomics in the past decade. One of the earliest isobaric tags-based technologies is TMT (tandem mass tags), which relies on the comparison of the unique "reporter ions" intensities for relative peptide/protein quantification. This differential labeling approach has evolved over time with respect to its multiplexing capability, i.e., from just 2 samples (TMTduplex) to 10 samples (TMT10plex) and a nowadays of up to 16 samples (TMTpro 16plex). Here, we describe a straightforward protocol to perform relatively deep proteome quantitative analyses using TMT10plex.


Assuntos
Proteínas/análise , Proteoma , Proteômica , Espectrometria de Massas em Tandem , Células HeLa , Humanos , Projetos de Pesquisa
19.
Methods Mol Biol ; 2228: 133-144, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950488

RESUMO

Isobaric peptide termini labeling (IPTL) is an approach for quantitative proteomics based on crosswise isotopic labeling of peptides at the N- and C-terminus. The labeling reagents are chosen in isotopic variations that the resulting mass of all labels per peptide is isobaric, but the individual label on each peptide terminus is different. Therefore, the quantitative difference of the peptide signal can be determined by the fragment ions of the corresponding MS2 spectra. Here, we describe an approach for triplex-IPTL to allow the comparison of three proteomes. This approach is based on digestion of the proteins by endoproteinase Lys-C, followed by three combinations of selective dimethylation of the peptide N-termini and subsequent dimethylation of the lysine residues at the C-termini. Data analysis is performed using Mascot for database searches and the freely available software package IsobariQ for quantification.


Assuntos
Marcação por Isótopo , Proteínas/análise , Proteoma , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Humanos , Projetos de Pesquisa
20.
Methods Mol Biol ; 2228: 145-157, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950489

RESUMO

Targeted proteomics represents an efficient method to quantify proteins of interest with high sensitivity and accuracy. Targeted approaches were first established for triple quadrupole instruments, but the emergence of hybrid instruments allowing for high-resolution and accurate-mass measurements of MS/MS fragment ions enabled the development of parallel reaction monitoring (PRM). In PRM analysis, specific peptides are measured as representatives of proteins in complex samples, with the full product ion spectra being acquired, allowing for identification and quantification of the peptides. Ideally, corresponding stable isotope-labeled peptides are spiked into the analyzed samples to account for technical variation and enhance the precision. Here, we describe the development of a PRM assay including the selection of appropriate peptides that fulfill the criteria to serve as unique surrogates of the targeted proteins. We depict the sequential steps of method development and the generation of calibration curves. Furthermore, we present the open-access tool CalibraCurve for the determination of the linear concentration ranges and limits of quantification (LOQ).


Assuntos
Marcação por Isótopo , Proteínas/análise , Proteoma , Proteômica , Espectrometria de Massas em Tandem , Animais , Calibragem , Humanos , Marcação por Isótopo/normas , Limite de Detecção , Proteômica/normas , Padrões de Referência , Projetos de Pesquisa , Espectrometria de Massas em Tandem/normas
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