Amounts Of Specific Proteins Research Articles

Revisiting ELISA with in situ amplification of biomarkers to boost its sensitivity

The enzyme-linked immunosorbent assay (ELISA) stands as a pivotal instrument in diagnostics and biomedical research, widely used for the detection and quantification of specific proteins associated with diseases. Despite its critical role, ELISA's ability to identify low-concentration proteins remains a significant challenge, hindering its effectiveness in early diagnostics, an area of growing importance. This study introduces a novel approach termed nucleic acid-templated amplification (NATA) to enhance ELISA's sensitivity without significantly modifying the common practices of the present ELISA. Drawing inspiration from viral proliferation mechanisms, our approach integrates a cell-free protein synthesis step using DNA sequences that encode the target biomarkers. This approach enables an in situ 'amplification' of target proteins during ELISA, boosting their concentrations to detectable levels. Our results demonstrate a substantial enhancement in sensitivity, with ELISA now being able to detect protein biomarkers at femtomolar concentrations—approaching the sensitivity range typically reserved for PCR in nucleic acid analysis. This breakthrough not only broadens ELISA’s applicability in early disease diagnosis and monitoring therapeutic interventions but also marks a significant advancement in protein biomarker detection technology. With its modular design, our method also offers high versatility and sensitivity, offering an advanced approach for protein detection in clinical diagnostics and research.

Antibody Design for the Quantification of Photosynthetic Proteins and Their Isoforms.

Antibodies are a valuable research tool, with uses including detection and quantification of specific proteins. By using peptide fragments to raise antibodies, they can be designed to differentiate between structurally similar proteins, or to bind conserved motifs in divergent proteins. Peptide sequence selection and antibody validation are crucial to ensure reliable results from antibody-based experiments. This chapter describes the steps for the identification of peptide sequences to produce protein- or isoform-specific antibodies using recombinant technologies as well as the subsequent validation of such antibodies. The photosynthetic protein Rubisco activase is used as a case study to explain the various steps involved and key aspects to take into consideration.

Dehydroepiandrosterone modulates the PTEN/PI3K/AKT signaling pathway to alleviate 4-vinylcyclohexene diepoxide-induced premature ovarian insufficiency in rats.

Dehydroepiandrosterone (DHEA) is frequently integrated as an adjuvant in over a quarter of controlled ovarian hyperstimulation (COH) protocols, despite the ongoing debate regarding its impact. This study aimed to evaluate the efficacy and mechanism of action of DHEA on ovarian follicular development and ovarian response in rats with varying ovarian reserves. The study involved 75 rats categorized into 15 distinct groups. The ovarian tissues of rats in both the normal ovarian reserve group and the premature ovarian insufficiency (POI) group, induced by 4-vinylcyclohexene diepoxide (VCD) injection, were subjected to histomorphological and biochemical analyses following the administration of DHEA, either alone or in combination with COH. Follicle counting was performed on histological sections obtained from various tissues. Serum concentrations of anti-Müllerian hormone (AMH) and the quantification of specific proteins in ovarian tissue, including phosphatase and tensin homolog of chromosome 10 (PTEN), phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (pAKT), cyclooxygenase 2 (COX-2), caspase-3, as well as assessments of total antioxidant status and total oxidant status, were conducted employing the ELISA method. The impact of DHEA exhibited variability based on ovarian reserve. In the POI model, DHEA augmented follicular development and ovarian response to the COH protocol by upregulating the PTEN/PI3K/AKT signaling pathway, mitigating apoptosis, inflammation, and oxidative stress, contrary to its effects in the normal ovarian reserve group. In conclusion, it has been determined that DHEA may exert beneficial effects on ovarian stimulation response by enhancing the initiation of primordial follicles and supporting antral follicle populations.

Development of Tier 2 LC-MRM-MS protein quantification methods for liquid biopsies

In the pursuit of personalized diagnostics and tailored treatments, quantitative protein tests contribute to a more precise definition of health and disease. The development of new quantitative protein tests should be driven by an unmet clinical need and performed in a collaborative effort that involves all stakeholders. With regard to the analytical part, mass spectrometry (MS)-based platforms are an excellent tool for quantification of specific proteins in body fluids, for example focused on cancer. The obtained readouts have great potential in determining tumor aggressiveness to facilitate treatment decisions, and can furthermore be used to monitor patient response. Internationally standardized TNM classifications of malignant tumors are beneficial for diagnosis, however treatment outcome and survival of cancer patients is poorly predicted. To this end, the importance of the tumor microenvironment has endorsed the introduction of the tumor-stroma ratio as a prognostic parameter in solid primary tumor types. Currently, the stromal content of tumor tissues is determined via routine diagnostic pathology slides. With the development of liquid chromatography (LC)-MS methods we aim at quantification of tumor-stroma specific proteins in body fluids. In this mini-review the analytical aspect of this developmental trajectory is further detailed.

Quantitative Detection of Protein Splice Variants by Selected Reaction Monitoring (SRM) Mass Spectrometry.

Molecular diversification of the cellular proteome through alternative splicing has emerged as an important biological principle. However, the lack of tools to specifically detect and quantify proteoforms (Smith et al., Nat Methods 10:186-187, 2013) is a major impediment to functional studies. Recently, biological mass spectrometry (MS) has undergone impressive advances (Mann, Nat Rev Mol Cell Biol 17:678, 2016), including the generation of a highly diverse set of biological applications (Aebersold and Mann, Nature 537:347-355, 2016), and has demonstrated to be an essential tool to address many biological questions (Savitski et al., Science 346:1255784, 2014; Rinner et al., Nat Methods 5:315-318, 2008). In particular, targeted LC-MS, with its high selectivity and specificity, is ideally suited for the precise and sensitive quantification of specific proteins and their proteoforms (Picotti and Aebersold, Nat Methods 9:555-566, 2012). We describe in detail the application of this workflow applied to dissect the molecular diversity of the synaptic adhesion proteins and their splicing-derived proteoforms (Schreiner et al., Elife 4:e07794, 2015).

PSV-B-19 Dietary choline supplementation increases transmethylation through the one-carbon cycle in overweight cats

Abstract The objective of this study was to determine the dose-response relationship between dietary choline intake and one-carbon cycle activity in overweight cats at maintenance energy requirements. This study was approved by the University of Guelph Animal Care Committee (AUP#4118). Overweight (body condition score:≥6/9) adult male neutered cats (n = 14) received each of five choline chloride doses individually for three weeks in a 5x5 latin square design. Doses were based on body weight (BW) and the daily recommended allowance (RA) for choline for adult cats (63 mg/kg BW0.67). Doses were: 2xRA (126 mg/kg BW0.67), 4xRA (252 mg/kg BW0.67), 6xRA (378 mg/kg BW0.67), 8xRA (504 mg/kg BW0.67), and control (no additional choline). Choline was administered onto the commercial extruded cat food (3620 mg choline/kg diet), given once a day at maintenance energy requirements (130 kcal/kg BW0.4). Fasted serum samples were analyzed for metabolomics through quantitative nuclear magnetic resonance (NMR) spectroscopy and direct infusion mass spectrometry (DI-MS) at the end of each period. Data was analyzed using proc GLIMMIX in SAS, with group and period as the random effects, and dose as the fixed effect. Statistical significance was considered at P < 0.05. Tukey’s post-hoc test was performed to assess multiple comparisons. Choline at 6x and 8x RA appears to increase transmethylation through the one-carbon cycle, as suggested by increased serum betaine, dimethylglycine (DMG) and methionine (P < 0.05). Carnitine, methylated by s-adenosylmethionine (SAM), increased (P < 0.05). Serine and threonine, precursors for the biosynthesis of homocysteine, appear to have been spared (P < 0.05). Supplemental dietary choline (6x and 8xRA) may be beneficial for overweight cats, as it may increase activity of the one-carbon cycle and methyl status; which are necessary for hepatic function. To better understand the impact of choline on methylation reactions, the quantification of specific proteins, gene expression and/or conversion rates is required.

Toxigence of anthrax vaccine strains

Abstract. The article presents the results of the study of strains of anthrax and anthrax bacilli on the formation of toxins. We found that anthrax vaccine strains actively produce exotoxins to the culture fluid. The amount of specific protein is different under the same incubation conditions and depends on the individual characteristics of the population of microorganisms, because of this, different titers of the toxin are registered. Strain B. anthracis K-79 Z (vaccine) with the same number of planting microbial cells and growing on the same nutrient medium and temperature produces two orders of magnitude more exotoxin than strains of B. anthracis Tsenkovsky II IVM 92 Z (virulent), B. anthracis Stern 34F2, (vaccine), B. anthracis 55 (vaccine), B. anthracis SB (vaccine), B. anthracis Tsenkovsky I (vaccine, а pathogenic). The amount of exotoxin may change as the pH of the medium changes. The activity of exotoxin production when the pH changes depends on the characteristics of the anthrax strain. Key words : anthrax, exotoxin, exotoxin production

An Efficient Combination among sMRI, CSF, Cognitive Score, and APOE ε4 Biomarkers for Classification of AD and MCI Using Extreme Learning Machine.

Alzheimer's disease (AD) is the most common cause of dementia and a progressive neurodegenerative condition, characterized by a decline in cognitive function. Symptoms usually appear gradually and worsen over time, becoming severe enough to interfere with individual daily tasks. Thus, the accurate diagnosis of both AD and the prodromal stage (i.e., mild cognitive impairment (MCI)) is crucial for timely treatment. As AD is inherently dynamic, the relationship between AD indicators is unclear and varies over time. To address this issue, we first aimed at investigating differences in atrophic patterns between individuals with AD and MCI and healthy controls (HCs). Then we utilized multiple biomarkers, along with filter- and wrapper-based feature selection and an extreme learning machine- (ELM-) based approach, with 10-fold cross-validation for classification. Increasing efforts are focusing on the use of multiple biomarkers, which can be useful for the diagnosis of AD and MCI. However, optimum combinations have yet to be identified and most multimodal analyses use only volumetric measures obtained from magnetic resonance imaging (MRI). Anatomical structural MRI (sMRI) measures have also so far mostly been used separately. The full possibilities of using anatomical MRI for AD detection have thus yet to be explored. In this study, three measures (cortical thickness, surface area, and gray matter volume), obtained from sMRI through preprocessing for brain atrophy measurements; cerebrospinal fluid (CSF), for quantification of specific proteins; cognitive score, as a measure of cognitive performance; and APOE ε4 allele status were utilized. Our results show that a combination of specific biomarkers performs well, with accuracies of 97.31% for classifying AD vs. HC, 91.72% for MCI vs. HC, 87.91% for MCI vs. AD, and 83.38% for MCIs vs. MCIc, respectively, when evaluated using the proposed algorithm. Meanwhile, the areas under the curve (AUC) from the receiver operating characteristic (ROC) curves combining multiple biomarkers provided better classification performance. The proposed features combination and selection algorithm effectively classified AD and MCI, and MCIs vs. MCIc, the most challenging classification task, and therefore could increase the accuracy of AD classification in clinical practice. Furthermore, we compared the performance of the proposed method with SVM classifiers, using a cross-validation method with Alzheimer's Disease Neuroimaging Initiative (ADNI) datasets.

Prospect and challenge of detecting dynamic gene copy number increases in stem cells by whole genome sequencing

Gene amplification is an evolutionarily well-conserved and highly efficient mechanism to increase the amount of specific proteins. In humans, gene amplification is a hallmark of cancer and has recently been found during stem cell differentiation. Amplifications in stem cells are restricted to specific tissue areas and time windows, rendering their detection difficult. Here, we report on the performance of deep WGS sequencing (average 82-fold depth of coverage) on the BGISEQ with nanoball technology to detect amplifications in human mesenchymal and neural stem cells. As reference technology, we applied array-based comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and qPCR. Using different in silico strategies for amplification detection, we analyzed the potential of WGS for amplification detection. Our results provide evidence that WGS accurately identifies changes of the copy number profiles in human stem cell differentiation. However, the identified changes are not in all cases consistent between WGS and aCGH. The results between WGS and the validation by qPCR were concordant in 83.3% of all tested 36 cases. In sum, both genome-wide techniques, aCGH and WGS, have unique advantages and specific challenges, calling for locus-specific confirmation by the low-throughput approaches qPCR or FISH.Key messagesWGS allows for the identification of dynamic copy number changes in human stem cells.Less stringent threshold setting is crucial for detection of copy number increase.Broad knowledge of dynamic copy number is pivotal to estimate stem cell capabilities.

Secretory Proteome of Brown Adipocytes in Response to cAMP-Mediated Thermogenic Activation.

Background: The secretory properties of brown adipose tissue are thought to contribute to the association between active brown fat and a healthy metabolic status. Although a few brown adipokines have been identified, a comprehensive knowledge of the brown adipose tissue secretome is lacking.Methods: Here, to examine the effects of thermogenic activation of brown adipocytes on protein secretion, we used isobaric tags for relative and absolute quantification (iTRAQ) analysis to determine how the secreted proteome of brown adipocytes (that detected in cell culture medium) differed in response to cAMP.Results: Our results indicated that 56 secreted proteins were up-regulated in response to cAMP. Of them, nearly half (29) corresponded to extracellular matrix components and regulators. Several previously known adipokines, were also detected. Unexpectedly, we also found five components of the complement system. Only 15 secreted proteins were down-regulated by cAMP; of them three were ECM-related and none was related to the complement system. We observed a partial concordance between the cAMP-regulated release of proteins (both from proteomics and from antibody-based quantification of specific proteins) and the cAMP-mediated regulation of their encoding transcript for the up-regulated secreted proteins. However, a stronger concordance was seen for the down-regulated secreted proteins.Conclusions: The present results highlight the need to investigate previously unrecognized processes such as the role of extracellular matrix in thermogenic activation-triggered brown fat remodeling, as well as the intriguing question of how brown adipocyte-secreted complement factors contribute to the signaling properties of active brown adipose tissue.

Targeted Proteomics for Multiplexed Verification of Markers of Colorectal Tumorigenesis

Targeted proteomic methods can accelerate the verification of multiple tumor marker candidates in large series of patient samples. We utilized the targeted approach known as selected/multiple reaction monitoring (S/MRM) to verify potential protein markers of colorectal adenoma identified by our group in previous transcriptomic and quantitative shotgun proteomic studies of a large cohort of precancerous colorectal lesions. We developed SRM assays to reproducibly detect and quantify 25 (62.5%) of the 40 selected proteins in an independent series of precancerous and cancerous tissue samples (19 adenoma/normal mucosa pairs; 17 adenocarcinoma/normal mucosa pairs). Twenty-three proteins were significantly up-regulated (n = 17) or downregulated (n = 6) in adenomas and/or adenocarcinomas, as compared with normal mucosa (linear fold changes ≥ ±1.3, adjusted p value <0.05). Most changes were observed in both tumor types (up-regulation of ANP32A, ANXA3, SORD, LDHA, LCN2, NCL, S100A11, SERPINB5, CDV3, OLFM4, and REG4; downregulation of ARF6 and PGM5), and a five-protein biomarker signature distinguished neoplastic tissue from normal mucosa with a maximum area under the receiver operating curve greater than 0.83. Other changes were specific for adenomas (PPA1 and PPA2 up-regulation; KCTD12 downregulation) or adenocarcinoma (ANP32B, G6PD, RCN1, and SET up-regulation; downregulated AKR1B1, APEX1, and PPA1). Some changes significantly correlated with a few patient- or tumor-related phenotypes. Twenty-two (96%) of the 23 proteins have a potential to be released from the tumors into the bloodstream, and their detectability in plasma has been previously reported. The proteins identified in this study expand the pool of biomarker candidates that can be used to develop a standardized precolonoscopy blood test for the early detection of colorectal tumors.

A label-free and enzyme-free ultra-sensitive transcription factors biosensor using DNA-templated copper nanoparticles as fluorescent indicator and hairpin DNA cascade reaction as signal amplifier

Detection and quantification of specific protein with ultralow concentration play a crucial role in biotechnological applications and biomedical diagnostics. In this paper, a label-free and enzyme-free amplified fluorescent biosensor has been developed for transcription factors detection based on AT-rich double-stranded DNA-templated copper nanoparticles (ds DNA/Cu NPs) and hairpin DNA cascade reaction. This strategy was demonstrated by using nuclear factor-kappa B p50 (NF-κB p50) and specific recognition sequences as a model case. In this assay, a triplex consists of double-stranded DNA containing NF-κB p50 specifically binding sequences and a special design single-stranded DNA (Trigger) which is able to activate the hairpin DNA cascade amplifier (HDCA). In the presence of NF-κB p50, the triplex became unstable since the target bound to the recognition sequences with strong affinity. The selective binding event confirmed that the Trigger was successfully released from the triplex and initiated HDCA to yield the product which could effectively template the formation of fluorescent Cu NPs. The experimental results revealed that the advanced strategy was ultra-sensitive for detecting NF-κB p50 in the concentration range from 0.1 to 1000 pM with a detection limit of 0.096 pM. In addition, the relative standard deviation was 4.08% in 3 repetitive assays of 500 pM NF-κB p50, which indicated that the reproducibility of this strategy was acceptable. Besides desirable sensitivity, the developed biosensor also showed high selectivity, cost-effective, and simplified operations. In addition, the proposed biosensing platform is versatile. By conjugating with various specific recognition units, it could hold considerable potential to sensitive and selective detect various DNA-binding proteins and might find wide applications in biomedical fields.

Spatial normalization of reverse phase protein array data.

Reverse phase protein arrays (RPPA) are an efficient, high-throughput, cost-effective method for the quantification of specific proteins in complex biological samples. The quality of RPPA data may be affected by various sources of error. One of these, spatial variation, is caused by uneven exposure of different parts of an RPPA slide to the reagents used in protein detection. We present a method for the determination and correction of systematic spatial variation in RPPA slides using positive control spots printed on each slide. The method uses a simple bi-linear interpolation technique to obtain a surface representing the spatial variation occurring across the dimensions of a slide. This surface is used to calculate correction factors that can normalize the relative protein concentrations of the samples on each slide. The adoption of the method results in increased agreement between technical and biological replicates of various tumor and cell-line derived samples. Further, in data from a study of the melanoma cell-line SKMEL-133, several slides that had previously been rejected because they had a coefficient of variation (CV) greater than 15%, are rescued by reduction of CV below this threshold in each case. The method is implemented in the R statistical programing language. It is compatible with MicroVigene and SuperCurve, packages commonly used in RPPA data analysis. The method is made available, along with suggestions for implementation, at http://bitbucket.org/rppa_preprocess/rppa_preprocess/src.

Accurate quantification of specific proteins of interest in single human blastocoels using targeted mass spectrometry

Accurate quantification of specific proteins of interest in single human blastocoels using targeted mass spectrometry

An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions.

Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-κB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.

Mass Spectrometry-based Workflow for Accurate Quantification of Escherichia coli Enzymes: How Proteomics Can Play a Key Role in Metabolic Engineering

Metabolic engineering aims to design high performance microbial strains producing compounds of interest. This requires systems-level understanding; genome-scale models have therefore been developed to predict metabolic fluxes. However, multi-omics data including genomics, transcriptomics, fluxomics, and proteomics may be required to model the metabolism of potential cell factories. Recent technological advances to quantitative proteomics have made mass spectrometry-based quantitative assays an interesting alternative to more traditional immuno-affinity based approaches. This has improved specificity and multiplexing capabilities. In this study, we developed a quantification workflow to analyze enzymes involved in central metabolism in Escherichia coli (E. coli). This workflow combined full-length isotopically labeled standards with selected reaction monitoring analysis. First, full-length (15)N labeled standards were produced and calibrated to ensure accurate measurements. Liquid chromatography conditions were then optimized for reproducibility and multiplexing capabilities over a single 30-min liquid chromatography-MS analysis. This workflow was used to accurately quantify 22 enzymes involved in E. coli central metabolism in a wild-type reference strain and two derived strains, optimized for higher NADPH production. In combination with measurements of metabolic fluxes, proteomics data can be used to assess different levels of regulation, in particular enzyme abundance and catalytic rate. This provides information that can be used to design specific strains used in biotechnology. In addition, accurate measurement of absolute enzyme concentrations is key to the development of predictive kinetic models in the context of metabolic engineering.

Novel calibration model maintenance strategy for solving the signal instability in quantitative liquid chromatography–mass spectrometry

In this contribution, a multiplicative effects model with a parameter accounting for the variations in overall sensitivity over time was proposed to reduce the effects of signal instability on quantitative results of LC–MS/MS. This method allows the use of calibration models constructed from large standard sets without having to repeat their measurement even though variations occur in sensitivity and baseline signal intensity. The performance of the proposed method was tested on two proof-of-concept model systems: the determination of the target peptide in two sets of peptide digests mixtures and the quantification of melamine and metronidazole in two sets of milk powder samples. Experimental results confirmed that multiplicative effects model could provide quite satisfactory concentration predictions for both systems with average relative predictive error values far lower than the corresponding values of various models investigated in this paper. Considering its capability in solving the problem of signal instability across samples and over time in LC–MS/MS assays and its implementation simplicity, it is expected that the multiplicative effects model can be developed and extended in many application areas such as the quantification of specific protein in cells and human plasma and other complex systems.

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.

Peptide Production and Decay Rates Affect the Quantitative Accuracy of Protein Cleavage Isotope Dilution Mass Spectrometry (PC-IDMS)

No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in protein cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent and postdigestion introduction of the SIL standards was related to differential decay experienced by the SIL peptide and the native peptide in each method. Given these results, we propose concurrent introduction of the SIL peptide is most appropriate, though not free from bias. Mathematical modeling of this method reveals that overestimation of protein quantities would still result when rapid peptide decay occurs and that this bias would be further exaggerated by slow proteolysis. We derive a simple equation to estimate the bias for each peptide based on the relative rates of production and decay. According to this equation, nearly half of the peptides evaluated here were estimated to have quantitative errors greater than 10% and in a few cases over 100%. We conclude that the instability of peptides can often significantly bias the protein quantities measured in protein cleavage isotope dilution mass spectrometry-based assays and suggest peptide stability be made a priority when selecting peptides to use for quantification.

Quantification of protein deposits on silicone hydrogel materials using stable-isotopic labeling and multiple reaction monitoring

This study was designed to use multiple reaction monitoring (MRM) for accurate quantification of contact lens protein deposits. Worn lenses used with a multipurpose disinfecting solution were collected after wear. Individual contact lenses were extracted and then digested with trypsin. MRM in conjunction with stable-isotope-labeled peptide standards was used for protein quantification. The results show that lysozyme was the major protein detected from both lens types. The amount of protein extracted from contact lenses was affected by the lens material. Except for keratin-1 (0.83 ± 0.61 vs 0.77 ± 0.20, p = 0.81) or proline rich protein-4 (0.11 ± 0.04 vs 0.15 ± 0.12, p = 0.97), the amounts of lysozyme, lactoferrin, or lipocalin-1 extracted from balafilcon A lenses (12.9 ± 9.01, 0.84 ± 0.50 or 2.06 ± 1.6, respectively) were significantly higher than that extracted from senofilcon A lenses (0.88 ± 0.13, 0.50 ± 0.10 or 0.27 ± 0.23, respectively) (p < 0.05). The amount of protein extracted from contact lenses was dependent on both the individual wearer and the contact lens material. This may have implications for the development of clinical responses during lens wear for different people and with different types of contact lenses. The use of MRM-MS is a powerful analytical tool for the quantification of specific proteins from single contact lenses after wear.