Protein Quantification Method Research Articles

Multiplexed Proteomic Analysis for Diagnosis and Screening of Five Primary Immunodeficiency Disorders From Dried Blood Spots.

Early detection of Primary Immunodeficiencies Disorders (PIDDs) is of paramount importance for effective treatment and disease management. Many PIDDs would be strong candidates for newborn screening (NBS) if robust screening methods could identify patients from dried blood spots (DBS) during the neonatal period. As majority of congenital PIDDs result in the reduction or absence of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool. Unfortunately, detection is often limited by the extremely low protein concentrations in blood cells and limited blood volume present in DBS. We have recently developed a robust novel method for quantification of low abundance proteins in DBS for PIDDs using peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-SRM). Here, we further generated a multiplexed Immuno-SRM panel for simultaneous screening of eight signature peptides representing five PIDD-specific and two cell-type specific proteins from DBS. In samples from 28 PIDD patients including two carriers, representing X-Linked Agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), X-Linked Chronic Granulomatous Disease (XL-CGD), DOCK8 Deficiency and ADA deficiency, peptides representing each disease are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. Also included in the multiplex panel are cell specific markers for platelets (CD42) and Natural Killer Cells (CD56). In patients with WAS, CD42 levels were found to be significantly reduced consistent with characteristic thrombocytopenia. A patient with WAS analyzed before and after bone marrow transplant showed normalized WAS protein and platelet CD42 after treatment highlighting the ability of immuno-SRM to monitor the effects of PIDD treatment. The assay was readily reproduced in two separate laboratories with similar analytical performance and complete agreement in patient diagnosis demonstrating the effective standardized methods. A high-throughput Immuno-SRM method screens PIDD-specific peptides in a 2.5-min runtime meeting high volume NBS workflow requirements was also demonstrated in this report. This high-throughput method returned identical results to the standard Immuno-SRM PIDD panel. Immuno-SRM peptide analysis represents a robust potential clinical diagnostic for identifying and studying PIDD patients from easily collected and shipped DBS and supports a significant potential for early PIDD diagnosis through newborn screening.

A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in the human liver.

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.

Blood-based detection of early-stage colorectal cancer using multiomics and machine learning.

66 Background: Despite population screening efforts, screening rates for colorectal cancer (CRC) remain suboptimal. A non-invasive, blood-based screening test with high sensitivity and specificity in early-stage disease should improve adherence and ultimately reduce mortality; however, tests based only on tumor-derived biomarkers have limited sensitivity. Here we used a multiomic, machine learning platform to discover, refine, and combine tumor- and immune-derived signals to develop a blood test for the detection of early-stage CRC. Methods: Samples from 591 participants enrolled in a prospective study including average-risk screening and case-control cohorts (NCT03688906) were included in this analysis (CRC: n = 43; colonoscopy-confirmed CRC-negative controls: n = 548). Participants with CRC were 60% male with a mean age of 63, and controls were 55% male with a mean age of 60. Stage distribution was 54% early (I/II) and 34% late (III/IV) with 11% unknown. Plasma was analyzed by whole-genome sequencing, bisulfite sequencing, and protein quantification methods. Computational methods were used to assess and infer the performance of individual and combined assays. Results: For colorectal adenocarcinoma, which represents ~95% of all CRCs, our multiomic test achieved a mean sensitivity of 92% in early stage (n = 17) and 84% in late stage (n = 11) at a specificity of 90%. Across all CRC pathological subtypes, our test achieved a mean sensitivity of 80% in early stage (n = 19) and 83% in late stage (n = 12) at a specificity of 90%; the test detected the single squamous cell carcinoma but missed both neuroendocrine tumors. Individual assays achieved a mean sensitivity of 50% in early stage and 66% in late stage at a specificity of 90%. Conclusions: In a prospective cohort, we demonstrated high sensitivity and specificity for early-stage adenocarcinoma by combining tumor- and immune-derived signals from cfDNA, epigenetic, and protein biomarkers. While most CRCs are adenocarcinomas, detection of all pathological subtypes is required to maximize sensitivity in a screening population. Further analysis of molecular and pathological subtypes, as well as the entire ~3000 patient cohort, is underway. Clinical trial information: NCT03688906.

A Cole‐Cole Dielectric Relaxation Analysis of Albumin and γ‐Globulins for Protein Quantification by Electrical Impedance Spectroscopy

AbstractOptimal serum protein concentrations are vital for normal body functioning. Affordable while accurate protein quantification methods with minimum processing requirements are needed for diagnosis of related diseases. The standard automated chemistry analyzer is limited by high installation and maintenance costs. This study proposes the use of electrical impedimetric spectroscopy (EIS) as an alternative to current methods. Its practical applicability was tested using albumin and γ‐globulin or their miscellanea in three different media; water, serum and tissue‐mimicking phantoms at 25 °C. Impedance measurements were taken between frequency f=0.10 MHz to 300 MHz by an impedance analyzer. A Cole‐Cole analysis was used to elucidate the stepwise variations in the dielectric parameters of the protein medium so as to obtain empirical dielectric parameter‐protein concentration relationships and their correlation coefficients R2. From the results, linear relationships between parameters and protein concentrations with high correlation coefficients over R2=0.90 were observed. Resistance to charge transfer Rct and characteristic frequency fc were significantly altered by changing protein concentrations as compared to bulk solution resistance Rs, relaxation time constant τ and shape factor α. The relationships developed would aid in monitoring changes in body fluid protein concentrations by EIS.

Novel Protein-oligonucleotide Conjugation Method Involving a High-affinity Capture HaloTag.

Highly sensitive quantitative protein profiling can play a key role in the early diagnosis of diseases, such as autoimmune diseases and cancer. We developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding, for quantifying protein molecules at a higher sensitivity than conventional protein quantification methods. This novel and efficient conjugation method can be used to prepare HaloTag-barcoded proteins using a click chemistry-based labeling technique. Here, we describe the preparation of protein-DNA complexes and detection of protein-protein interactions which can be used in a HaloTag protein barcode assay to detect an antibody. The protocol includes procedures for preparing the ligand-oligonucleotide complex, plasmid DNA preparation for protein expression, and preparation of the protein-oligonucleotide complex. The described click reaction-based protocols simplify the conventional amine-ester reaction methods which require additional steps for chromatography purification.

Carbon Nanotube-Based Electrochemical Biosensor for Label-Free Protein Detection.

There is a growing need for biosensors that are capable of efficiently and rapidly quantifying protein biomarkers, both in the biological research and clinical setting. While accurate methods for protein quantification exist, the current assays involve sophisticated techniques, take long to administer and often require highly trained personnel for execution and analysis. Herein, we explore the development of a label-free biosensor for the detection and quantification of a standard protein. The developed biosensors comprise carbon nanotubes (CNTs), a specific antibody and cellulose filtration paper. The change in electrical resistance of the CNT-based biosensor system was used to sense a standard protein, bovine serum albumin (BSA) as a proof-of-concept. The developed biosensors were found to have a limit of detection of 2.89 ng/mL, which is comparable to the performance of the typical ELISA method for BSA quantification. Additionally, the newly developed method takes no longer than 10 min to perform, greatly reducing the time of analysis compared to the traditional ELISA technique. Overall, we present a versatile, affordable, simplified and rapid biosensor device capable of providing great benefit to both biological research and clinical diagnostics.

New approach to evaluating the effects of a drug on protein complexes with quantitative proteomics, using the SILAC method and bioinformatic approach.

Protein-protein interactions (PPIs) lead the formation of protein complexes that perform biochemical reactions that maintain the living state of the living cell. Although therapeutic drugs should influence the formation of protein complexes in addition to PPI network, the methodology analyzing such influences remain to be developed. Here, we demonstrate that a new approach combining HPLC (high performance liquid chromatography) for separating protein complexes, and the SILAC (stable isotope labeling using amino acids in cell culture) method for relative protein quantification, enable us to identify the protein complexes influenced by a drug. We applied this approach to the analysis of thalidomide action on HepG2 cells, assessed the identified proteins by clustering data analyses, and assigned 135 novel protein complexes affected by the drug. We propose that this approach is applicable to elucidating the mechanisms of actions of other therapeutic drugs on the PPI network, and the formation of protein complexes.

Membrane Transporters in Human Parotid Gland-Targeted Proteomics Approach.

Salivary glands provide secretory functions, including secretion of xenobiotics and among them drugs. However, there is no published information about protein abundance of drug transporters measured using reliable protein quantification methods. Therefore, mRNA expression and absolute protein content of clinically relevant ABC (n = 6) and SLC (n = 15) family member transporters in the human parotid gland, using the qRT-PCR and liquid chromatography‒tandem mass spectrometry (LC−MS/MS) method, were studied. The abundance of nearly all measured proteins ranged between 0.04 and 0.45 pmol/mg (OCT3 > MRP1 > PEPT2 > MRP4 > MATE1 > BCRP). mRNAs of ABCB1, ABCC2, ABCC3, SLC10A1, SLC10A2, SLC22A1, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLCO1A2, SLCO1B1, SLCO1B3 and SLCO2B1 were not detected. The present study provides, for the first time, information about the protein abundance of membrane transporters in the human parotid gland, which could further be used to define salivary bidirectional transport (absorption and secretion) mechanisms of endogenous compounds and xenobiotics.

Targeted Proteomics as a Tool for Quantifying Urine-Based Biomarkers.

Mass spectrometry based proteomics approaches are routinely used to discover candidate biomarkers. These studies often use small number of samples to discover candidate proteins that are later validated on a large cohort of samples. Targeted proteomics has emerged as a powerful method for quantification of multiple proteins in complex biological matrix. Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) are two main methods of choice for quantifying and validating proteins across hundreds to thousands of samples. Over the years, many software tools have become available that enable the users to carry out the analysis. In this chapter, we describe selection of proteotypic peptides, sample preparation, generating a response curve, data acquisition and analysis of PRM data using Skyline software for targeted proteomics to quantify candidate markers in urine.

Certification and stability assessment of recombinant human growth hormone as a certified reference material for protein quantification

A certified reference material (CRM) for the quantification of protein, essential to manage quality control and quality assurance in protein-related works, has been developed. Amino acid analysis with conventional acid hydrolysis and isotope dilution HPLC-MS was used to establish an SI-traceable absolute protein quantification method using recombinant human growth hormone (hGH) as a model protein. The certification method was verified by comparative studies between 1) different methods of protein quantification based on microwave-assisted hydrolysis, and 2) different labs as part of the Asian Collaboration on Reference Material project with Japan, China, and Korea. Certification, evaluation of measurement uncertainty, homogeneity testing, and stability testing were carried out, after which the candidate CRM for hGH quantification was successfully certified with excellent agreement within the certified value in the two comparative studies. Although the quantification value of hGH by amino acid analysis showed good robustness in various conditions, results of intact protein analysis showed degradation profiles in temperatures higher than 4 °C. Consequently, storage and dissemination conditions should be set in accordance with stability tests. Based on the results, this method is believed to be suitable for accurate quantification of hGH. Additionally, it can also be used as a guide to preparation of CRM, and instructions for quality management of protein work for other similar proteins.

Royal jelly-derived proteins enhance proliferation and migration of human epidermal keratinocytes in an in vitro scratch wound model

BackgroundSkin injury is inevitable in daily life. In recent years, with the increasing morbidity of diseases such as diabetes and metabolic disorders, chronic wounds have become a considerable challenge in clinical practice. Royal jelly, reported to have multifarious biological and physiological properties, has been used as a remedy for a variety of wounds since ancient times. However, the active components and mechanisms underlying the wound-healing properties of royal jelly are still largely unknown.MethodsWater-soluble proteins of royal jelly were fractionated and investigated for the proliferative and migratory effects on human epidermal keratinocytes (HaCaT) in an in vitro wound healing model. The proteins present in bioactive fractions were characterised and quantified using Label-free protein quantification method. The potential functions of these proteins in biological systems were further analysed using bioinformatic tools.ResultsA protein fraction, mainly containing major royal jelly proteins 2 (MRJP2), MRJP3 and MRJP7, stimulated proliferative and migratory activities in HaCaT cells without visible cytotoxicity. It exerted the greatest effects on the growth of HaCaT cells in the first 48 h. Furthermore, when treated with this protein fraction, the closure rates of the in vitro scratch wound were significantly increased. Functional analysis indicated that MRJP2, MRJP3 and MRJP7 were associated with carbohydrate transport and metabolism.ConclusionsWe fractionated the water-soluble proteins of royal jelly and identified one fraction (Fraction 2) that induced both proliferative and migratory effects on a human epidermal keratinocyte cell line. Major royal jelly proteins (MRJP2, MRJP3 and/or MRJP7) were speculated to possess potential wound-healing bioactivity. This is the first report that royal jelly may improve wound closure via MRJP-induced cellular proliferation and migration. These proteins may be valuable lead compounds for the development of novel wound healing medications. Our findings would facilitate better understanding of the wound repair mechanisms of royal jelly.

Glycoproteomics Method to Discover Reliable Biomarkers from Human Plasma of Lung Cancer Patients for MS‐based Clinical Studies

In clinical and diagnostic proteomics, it is important to discover significant biomarkers from biosamples. Thus, a reliable proteomics methodology is required for the development and standardization of an MS‐based protein identification and quantification method in biosamples. In particular, plasma is one of the most complex fluids in the human body and is commonly used in hospitals to diagnose disease. The purpose of this study was to develop a reliable and reproducible proteomics method using lung cancer plasma for biomarker discovery and diagnostic kits for screening. Glycoproteins are well‐known to be associated with diseases (especially cancers) so in this study glycoproteomics was used for discovering biomarkers from lung cancer plasma. Thirty‐five lung cancer plasma were pooled, and glycoproteins were separated using lectin affinity chromatography (LAC). After affinity selection, trypsin‐digestion, and deglycosylation with PNGase F, the resulting deglycosylated peptides were analyzed with nLC–MS/MS runs twice. The corresponding parent proteins were identified separately through two database search engines and then analyzed individually. The identified proteins from each set were compared, combined, and then categorized for analysis. The combined total identified number of proteins were about 50% increased than in a single run and some proteins seemed to be more reproducible and reliable biomarker candidates because they were always identified in every run. From this research, method‐optimized proteomics can be applied to biomarker discovery for diagnosis and prognosis of disease such as cancer for better MS‐based clinical studies. The identified plasma proteins from this research will also be lung cancer biomarker candidates and can be utilized for the development of in vitro lung cancer diagnostic kits.

Quantification of surfactant protein D (SPD) in human serum by liquid chromatography-mass spectrometry (LC-MS)

Quantification of intact proteins in complex biological matrices by liquid chromatography-mass spectrometry (LC-MS) is a promising analytical strategy but is technically challenging, notably for concentrations at or below the ng/mL level. Therefore, MS-based protein quantification is mostly based on measuring protein-specific peptides, so-called ‘surrogate peptides’, that are released through proteolysis. While quantitative protein bioanalysis based on peptide LC-MS is much more sensitive, not every peptide is suitable in this respect. For example, some peptides are too small to be unique for a protein while others are too large to be measured with sufficient sensitivity, so careful selection of appropriate peptides is essential. Here we present a validated LC-MS method for quantification of surfactant protein D (SPD) at clinically relevant levels between 5 and 500 ng/mL using 50 μL of serum. This method targets two SPD-specific peptides in the C-type lectin, ligand binding domain of the SPD protein. One of these peptides contains a methionine residue which would typically be avoided because of its unstable nature. Some quantitative methods do target methionine-containing peptides, and corresponding workflows feature an oxidation step at the peptide level using hydrogen peroxide (H2O2) to convert all methionine residues to more stable methionine sulfoxides. For our method, such a procedure was associated with peptide loss, hence we developed an oxidation procedure at the protein level using H2O2 to oxidize methionine residues and the enzyme catalase to quench excess H2O2. This procedure may be applicable to other quantitative methods based on a surrogate peptide-based approach and may potentially also be useful for MS-based workflows targeting intact proteins.

More than a Toxin: Protein Inventory of Clostridium tetani Toxoid Vaccines.

Clostridium tetani is the etiological agent of tetanus, a life-threatening bacterial infection. The most efficient protection strategy against tetanus is a vaccination with the C. tetani neurotoxin, which is inactivated by formaldehyde-crosslinking. Since we assumed that besides the tetanus toxin, other proteins of C. tetani may also be present in toxoid preparations, we analyzed commercially available vaccines from different countries in respect to their protein content using mass spectrometry. In total 991 proteins could be identified in all five analyzed vaccines, 206 proteins were common in all analyzed vaccines and 54 proteins from the 206 proteins were potential antigens. The additionally present proteins may contribute at least partially to protection against C. tetani infection by supporting the function of the vaccine against the devastating effects of the tetanus toxin indirectly. Two different label-free protein quantification methods were applied for an estimation of protein contents. Similar results were obtained with a Total Protein Approach (TPA)-based method and Protein Discoverer 2.2 software package based on the minora algorithm. Depending on the tetanus toxoid vaccine and the quantification method used, tetanus neurotoxin contributes between 14 and 76 % to the total C. tetani protein content and varying numbers of other C. tetani proteins were detected.

Label-free absolute protein quantification with data-independent acquisition

Despite data-independent acquisition (DIA) has been increasingly used for relative protein quantification, DIA-based label-free absolute quantification method has not been fully established. Here we present a novel DIA method using the TPA algorithm (DIA-TPA) for the absolute quantification of protein expressions in human liver microsomal and S9 samples. To validate this method, both data-dependent acquisition (DDA) and DIA experiments were conducted on 36 individual human liver microsome and S9 samples. The MS2-based DIA-TPA was able to quantify approximately twice as many proteins as the MS1-based DDA-TPA method, whereas protein concentrations determined by the two approaches were comparable. To evaluate the accuracy of the DIA-TPA method, we absolutely quantified carboxylesterase 1 concentrations in human liver S9 fractions using an established SILAC internal standard-based proteomic assay; the SILAC results were consistent with those obtained from DIA-TPA analysis. Finally, we employed a unique algorithm in DIA-TPA to distribute the MS signals from shared peptides to individual proteins or isoforms and successfully applied the method to the absolute quantification of several drug-metabolizing enzymes in human liver microsomes. In sum, the DIA-TPA method not only can absolutely quantify entire proteomes and specific proteins, but also has the capability quantifying proteins with shared peptides. SignificanceData independent acquisition (DIA) has emerged as a powerful approach for relative protein quantification at the whole proteome level. However, DIA-based label-free absolute protein quantification (APQ) method has not been fully established. In the present study, we present a novel DIA-based label-free APQ approach, named DIA-TPA, with the capability absolutely quantifying proteins with shared peptides. The method was validated by comparing the quantification results of DIA-TPA with that obtained from stable isotope-labeled internal standard-based proteomic assays.

Development of a first order derivative spectrophotometry method to rapidly quantify protein in the presence of chitosan and its application in protein encapsulation systems

A new protein quantification method based on first order derivative spectrophotometry was established to eliminate various interferences, mainly chitosan, to the utmost using bovine serum albumin (BSA) as a model food protein. Absorbance spectra of BSA solutions were recorded and their first order derivative calculated. The values of derivative absorbance at 288 nm were used to generate linear calibration curve of BSA. The new method was applied in entrapping BSA into chitosan-tripolyphosphate beads. A general calibration curve was established with a CI (width of 95% confidence interval of three repeat measurements of unknown samples) less than 0.0262 g/L and a LOQ (limit of quantification) of 0.11 g/L, showing excellent tolerance to various interferences, which was further verified by the good mass balance of BSA during encapsulation. Overall, the method successfully eliminated the interferences from chitosan and other factors to facilitate the measurement of protein in complicated environments.

A new SE-HPLC method for simultaneous quantification of proteins and main phenolic compounds from sunflower meal aqueous extracts.

The aim of this research was to develop a method for simultaneous quantification of proteins and main polyphenolic compounds extracted from oleaginous meal by aqueous media. Size exclusion chromatography with a Biosep column (exclusion range from 1 to 300kDa) and acetonitrile/water/formic acid (10:89.9:0.1 v/v) eluent at 0.6mLmin-1 yielded the most efficient separation of sunflower proteins and chlorogenic acid monoisomers (3-caffeoylquinic acid, 5-caffeoylquinic acid, and 4-caffeoylquinic acid). After a study of the stability of the extract components, the incorporation of a stabilization buffer (0.5molL-1 tris(hydroxymethyl)aminomethane-hydrochloric acid/1.0molL-1 sodium chloride at pH7) was proposed to avoid polyphenol-protein interactions and/or isomeric transformation. The use of 214nm as the wavelength for protein quantification was also included to minimize the effect of interference from polyphenol-protein interactions on the quantification. Under the used experimental conditions, the protein and chlorogenic acid monoisomer signals remained stable during 300min at 20°C (95-125% of the starting value). The developed method was validated and parameters such as specificity, sensitivity, precision, and accuracy were determined. The results from size exclusion chromatography correlated well with the results of protein determination by the reference Kjeldahl method. The proposed method was successfully applied for rapeseed extract analysis making simultaneous quantification of proteins and major rapeseed polyphenols (sinapine and sinapic acid) possible. Graphical abstract.

Comparative Analysis of Protein Quantification Methods for the Rapid Determination of Protein Loading in Liposomal Formulations.

Advances in manufacturing processes provide the ability for the high throughput production of liposomes containing a range of moieties, from small molecules to large biologicals (including proteins and nucleic acids for prophylactic and therapeutic applications). Whilst rapid quantification methods for small molecules are generally well established, the ability to rapidly quantify liposomal entrapment of proteins is limited. Indeed, most standard protein quantification techniques (including the BCA assay and Reverse phase-high performance liquid chromatography (RP-HPLC)) measure protein encapsulation indirectly, by measuring the amount of non-incorporated drug, and subtracting from the initial amount of protein added. However, this can give inaccurate and misrepresentative results. To address this, we have developed a range of methods to directly quantify protein entrapment within liposomes. The encapsulation efficiency within neutral, anionic and cationic liposome formulations was determined by three techniques; BCA assay, RP-HPLC and HPLC coupled to an evaporative light scattering detector, (HPLC-ELSD). All three methods are reliable for the quantification of protein, with linear responses and correlation coefficients of 0.99, and LOQ for all three methods being less than 10 µg/mL. Here within, we provide three methods for the rapid and robust quantification of protein loading within liposomal (and other bilayer) vesicle systems.

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples.

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

Increasing the amount of phosphoric acid enhances the suitability of Bradford assay for proteomic research.

The Bradford assay is one of the most commonly used methods for protein quantification. However, in proteomic research, the lysis buffer generally used for dissolving proteins can cause some interference to the assay. The dye reagent of classical Bradford assay contains 8.50% (w/v) phosphoric acid, which is an important factor relating to the color yield of the assay. In this study, the phosphoric acid content in dye reagent was increased to 9.35% (w/v), 10.20% (w/v), and 11.05% (w/v) to evaluate the changes of interference and the effects of lysis buffer on the interaction between proteins and dye reagent. Results show that lysis buffer not only causes background interference but also affects the protein-dye chromogenic process. Analysis of different components in the lysis buffer showed that carrier ampholyte is the main factor that introduces interference to the Bradford assay. Detergents are well-known interfering compounds in the Bradford assay, but CHAPS and octyl b-D-glucopyranoside only cause slight interference. When the amount of phosphoric acid was increased from 8.50%(w/v) to 10.20% (w/v), the sensitivity of the Bradford assay to proteins in lysis buffer was increased, and the interference delivered by lysis buffer was considerably reduced.