Sample Digestion Research Articles

Phosphoproteomics After Guanidinium Thiocyanate Extraction of Tissue Biopsies.

Proteogenomic analysis is emerging as an advantageous tool to assist personalized therapy decisions in clinical health care and integrates complementary information from the genome, transcriptome, and (phospho)proteome. A prerequisite for such analysis is a workflow for the simultaneous isolation of DNA, RNA, and protein from a single sample that does not compromise the different biological molecules and their examination. Focusing on the phosphoproteomic aspect of this workflow, we here provide detailed information on our protocol, which is based on commonly used acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction with RNA-Bee. We describe the necessary steps for biopsy collection, cryoprocessing, and protein extraction. We further share our practice on protein digestion and cleanup of small samples (200μg protein) and describe settings for automated IMAC-based phosphopeptide enrichment with the AssayMAP Bravo platform.

Protein-Protein Interaction Network Mapping by Affinity Purification Cross-Linking Mass Spectrometry (AP-XL-MS) based Proteomics.

Protein-protein interactions (PPIs) are the physical interactions formed among proteins. These interactions are primarily functional, i.e., they arise from specific biomolecular events, and each interaction interface serves a specific purpose. A significant number of methods have been developed for protein interactions in the field of proteomics in the last decade. Advanced mass spectrometry technology significantly contributed to the development of these methods. The rapid advancement of groundbreaking MS technology has greatly aided themapping ofprotein interaction from large-data sets comprehensively. This chapter describes the affinity purification (AP) mass spectrometry (MS)-based methods combined with chemical cross-linking (XL) of protein complexes. This chapter includes sample preparation methods involving cell culture, cell treatments with ligands, drugs, and cross-linkers, protein extractions, affinity purification, sodium dodecyl sulfate (SDS) polyacrylamide gel separation, in-solution or in-gel digestion, liquid-chromatography, and mass spectrometry analysis of samples (LC-MS/MS). Application of a cleavable cross-linker, dual cleavable cross-linking technology (DUCCT) in combination with the affinity purification (AP) method has also been described. Methods for data analysis using unmodified and cross-linked peptide analysis arediscussed.

METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF CLASS-I ELEMENTAL IMPURITIES IN PROPOFOL EMULSION USING ICP-MS

Elemental impurities are the traces of metals that can be found in finished drug products which may enter through various sources like excipients, and active pharmaceutical ingredients etc., their toxicity is to be estimated as they may pose serious toxicological effects to patients. The aim of this work is to estimate Class-I elements (Cd, Pb, As, Hg) simultaneously in Propofol 10 mg/mL Emulsion for Injection or Infusion using modern technique Inductively coupled plasma mass spectrometry, employing microwave-assisted digestion of sample. The developed method was validated for various parameters according to ICHQ2(R1) to prove that the developed method is fit for the required purpose and can be adaptable regularly. Validation results showed that the method was linear over the range of LOQ to 250%, with a good linearity correlation factor of R≥0.99, the method was found to be robust against the deliberate changes in sample preparation and uptake speed.

Assessment of level of heavy metals in cosmetics

Background: Toxic metals in cosmetic products can lead to serious health problems among consumers. Skin lightening cosmetics are popular among women who may be unaware of prevalence of toxic metals in such products. Purpose: This study assessed the content of toxic metals in cosmetic products imported into Botswana. There are currently no regulations in Botswana governing maximum contaminants limits for contaminants such as metal ions in cosmetic products. Research Design: Sample analysis was carried out using inductively coupled plasma optical emission spectrometry after microwave-assisted acid sample digestion. Results: Total concentrations of metals were in the order Pb > Ni > Cr > As. The concentrations of metals analysed were in the range of 45.75–193.60; 2.99–9.50; 3.32–7.41 and 1.95–4.52 mg/kg for Pb, Ni, Cr and As respectively. The concentrations of Pb in all 14 samples exceeded the maximum impurity level of 10 mg/kg in cosmetics as per the United States Food and Drug Administration (US FDA). Concentrations of Ni, As and Cr were also higher than the set US FDA limits in some cosmetic samples under study. A human health risk assessment was conducted using hazard quotient, average daily intake and health risk index. A hazard quotient of greater than one was determined for Pb (HQ ∼ 177.0–749.2) in all 14 cosmetic samples investigated indicating potential adverse effects to human health. Conclusion: Overall results of total concentration and health risk assessment indicate that cosmetic products imported into Botswana are harmful to consumers. Therefore, quality control measures should be enforced to ensure metal concentrations in facial cosmetic products do not exceed regulatory limits.

Effects of water bath digestion and DEENA pretreatment on mercury content detection in soil

There are many sample preparation methods for heavy metal detection of soil samples, but each has its advantages and disadvantages. Based on the above two pretreatment methods, the detection limit, precision, accuracy and actual soil sample measurement value of mercury detection in soil by atomic fluorescence were compared with atomic fluorescence, and the results showed that the automatic digestion and pretreatment system (DEENA) could digest soil samples to meet the requirements of GB/T 22105.1-2008 standard. Moreover, there was no significant difference between the two digestion methods on the detection results of mercury in soil, and the automatic digestion and preparation system (DEENA) was convenient, operable and highly automated, which was more advantageous for sample digestion.

From mouth to gut: microfluidic in vitro simulation of human gastro-intestinal digestion and intestinal permeability.

Reproducible in vitro studies of bioaccessibility, intestinal absorption, and bioavailability are key to the successful development of novel food ingredients or drugs intended for oral administration. There is currently a lack of methods that offer the finesse required to study these parameters for valuable molecules typically found in small volumes - as is the case of nanomaterials, which are often used to carry and protect bioactives. Here, we describe a modular microfluidic-based platform for total simulation of the human gastro-intestinal tract. Digestion-chips and cell-based gut-chips were fabricated from PDMS by soft lithography. On-chip digestion was validated using a fluorescently labelled casein derivative, which followed typical Michaelis-Menten kinetics and showed temporal resolution and good agreement with well-established bench-top protocols. Irreversible inhibition of serine proteases using Pefabloc® SC and a 1 : 6 dilution was sufficient to mitigate the cytotoxicity of simulated digestion fluids. Caco-2/HT29-MTX co-cultures were grown on-chip under a continuous flow for 7 days to obtain a differentiated cell monolayer forming a 3D villi-like epithelium with clear tight junction formation, and with an apparent permeability (Papp) of Lucifer Yellow closely approximating values reported ex vivo (3.7 × 10-6 ± 1.4 × 10-6vs. 4.0 × 10-6 ± 2.2 × 10-6). Digesta from the digestion-chips were flowed through the gut-chip, demonstrating the capacity to study sample digestion and intestinal permeability in a single microfluidic platform holding great promise for use in pharmacokinetic studies.

Effect of ethanol on the diatom test using nitric acid or sodium hypochlorite

ObjectiveTo study the quantitative effect of ethanol on the diatom test for water and lung samples. Materials and methodsIn experiment 1, we tested 20 water samples taken from natural water areas. In experiments 1–1 and 1–2, each sample was digested with sodium hypochlorite (NaClO) solution (Purelox) and fuming nitric acid (HNO3), respectively. In Experiments 1–3 and 1–4, each sample was added to a lung sample containing few diatoms and digested with NaClO and HNO3, respectively. In Experiment 2, eight lung samples containing diatoms were digested with NaClO. Then, each digested sample was divided into two portions; one portion was washed with ethanol before being washed with water, and the other was washed only with water. After base-2 logarithmic transformation, the counts from the methods with and without ethanol wash were compared with a paired t-test. ResultsIn experiments 1–1, 1–2, 1–3, 1–4, and 2, the geometric means of the ratios derived from the two methods (with/without ethanol) were 0.70 (95 % confidence interval [CI]: 0.65–0.77, P < 0.001), 0.83 (95 % CI: 0.73–0.93, P = 0.005), 3.00 (95 % CI: 2.31–3.91, P < 0.001), 0.91 (95 % CI: 0.79–1.04, P = 0.164), and 3.06 (95 % CI: 2.28–4.41, P < 0.001), respectively. ConclusionOur experiments suggest that ethanol would be useless in diatom tests of water samples or in the conventional (HNO3) digestion of lung samples. However, ethanol is essential for the NaClO digestion of lung samples and could also be useful for other alternative methods of lung samples.

Determination of thallium in vegetative plant leaves near industrial areas by high-resolution continuum source electrothermal atomic absorption spectrometry after salt induced cloud point extraction

A rapid analytical procedure is described for the separation and pre-concentration of ultra trace level thallium from the microwave assisted nitric acid digests of plant samples and determination using high resolution continuum source electrothermal atomic absorption spectrometry (HR-CS-ETAAS). The method is based on the extraction of ionic thallium spices formed in-situ in the nitric acid digests of plant leaves with Triton X-100 micelles, in presence of HCl and NaCl. The hydrophobic solubilising sites of Triton X-100 micelles are used for the pre-concentration of hydrophobic species of thallium from bulk aqueous phase into a small surfactant rich phase. The main parameters affecting the extraction process are evaluated and optimized. A rapid temperature program completed within 15 s is used for the determination of thallium using a permanent ZrIr modifier. Under the optimized conditions, the pre-concentration factor and limit of detection are 25 and 0.05 ng mL−1, respectively. A characteristic mass of 12 pg is obtained on measuring the integrated absorbance with central pixel ±1. The recoveries are in the range of 95–99% at 1 ng mL−1 with 2–3% relative standard deviation. The accuracy of the method is verified by analyzing certified reference materials such as BCR 060 aquatic plant and NIST 1572 citrus leaves. The obtained values are in good agreement with the certified values. The procedure is also applied to real samples such as spinach, cabbage, aquatic, and tobacco plant leaves collected near thermal power plants and industrial areas.

Effects of In Vitro Digestion on the Antioxidant Activity of Three Phenolic Extracts from Olive Mill Wastewaters.

The aim of this study was to assess the impact of in vitro digestion on the antioxidant activity of three extracts rich in phenols (two purified organic extracts (A20, A21) and one powdered extract stabilized with maltodextrins (SP)) obtained from olive mill wastewaters (OMWW). The content and composition of phenols and antioxidant activity was determined before and after in vitro digestion. The phenol content of the A20 and A21 samples were higher (&gt;75%) than that of the SP sample before in vitro digestion. After the entire in vitro digestion, 89.3, 76.9, and 50% loss of phenols was found in A20, A21 and SP, respectively. ABTS•+ and ORAC values decreased during in vitro digestion of A20 and A21 samples, while they remained almost constant in SP. IC50 increased during digestion of A20 and A21, evidencing a loss of antioxidant capacity after the intestinal phase; an opposite IC50 trend was noted in SP, confirming the protective role of maltodextrins. For these reasons, SP represents a promising formulation to be used in the food field.

Managing health risks in urban agriculture: The effect of vegetable washing for reducing exposure to metal contaminants

A common, yet poorly evaluated, advice to remove contaminants from urban vegetables is to wash the produce before consumption. This study is based on 63 samples of chard, kale, lettuce and parsley that have grown near a heavily trafficked road in the third largest city in Sweden, with one portion of each sample being analysed without first being washed, and the other portion being subjected to common household washing. Concentrations of 71 elements were analysed by ICP-SFMS after a sample digestion that dissolves both the plant tissues and all potentially adhering particles. The results show that the washing effect, or the fraction removed upon washing, varies significantly between elements: from approximately 0 % for K to 68 % for the ∑REEs. Considering traditional metal contaminants, the efficiency decreased from Pb (on average 56 % lost) to Co (56 %) > Cr (55 %) > As (45 %) > Sb (35 %) > Ni (33 %) > Cu (13 %) > Zn (7 %) > Cd (7 %), and Ba (5 %). A clear negative correlation between the washing effect and the different elements' bioconcentration factors shows that the elements' accessibility for plant uptake is a key controlling factor for the degree to which they are removed upon washing. Based on the average washing efficiencies seen in this study, the average daily intake of Pb would increase by 130 % if vegetables are not washed prior to consumption. For the other contaminant metals this increase corresponds to 126 % (Co), 121 % (Cr), 82 % (As), 55 % (Sb), 50 % (Ni), 16 % (Cu), 8 % (Zn), 7 % (Cd) and 5 % (Ba). The advice to wash vegetables is therefore, for many elements, highly motivated for reducing exposure and health risks. For elements which are only slightly reduced when the vegetables are washed, however, advising should rather focus on reducing levels of contamination in the soil itself.

Do predator (Mystus gulio) and prey (Penaeus monodon) have differential response against heatwaves? Unveiling through oxidative stress biomarkers and thermal tolerance estimation

Extreme climatic events such as heatwaves are anticipated to intensify in future and impose additional thermal stress to aquatic animals. Knowledge regarding an organism's thermal tolerance or sensitivity is therefore important in determining the effects of fluctuating water temperature on physiological responses. Thus, thermal tolerance tests can serve as a first step in understanding the present and future effects of climate warming. Climatic variability will alter prey-predator attributes differentially and impact their subsequent interactions. The key objective of this study was to compare and decode the stress responses, resistance and vulnerability of two economically important species from Sundarbans estuarine system- Penaeus monodon (prey) and Mystus gulio (predator) subjected to acute thermal challenges such as sudden heatwaves. Both the species were subjected to an increasing thermal ramp of 1°C h−1 from 22°C to 42°C. Organisms were observed continuously throughout the ramping period and changes in the locomotory behaviour were followed until their loss of equilibrium. The digestive tissue samples were dissected out from both M. gulio and P. monodon at every 2°C and also after a recovery period of 48 h. The SOD, CAT, GST, LPO were measured and integrated biomarker response (IBR) was analysed. The results from thermal tolerance maxima estimation, biomarker study, IBR responses indicated more intense stress response in fish M. gulio whereas recovery potential was greater in shrimp P. monodon. Our findings corroborate the ‘trophic sensitivity hypothesis’ which advocates predators to be less tolerant in aggravated environmental stress than their prey.

Investigation of accumulation ability of crops in point of dynamics of residual quantities if copper-containing pesticides

Relevance. Study of pesticide residues is an integral part of registration tests. It is present a particular interest in terms of hygienic safety of eating vegetable crops grown with use of protective agents.Methods. The analysis procedure includes an approach developed by the authors, based on microwave digestion of samples after cryo-destruction, followed by analysis by atomic absorption spectrometry with electrothermal atomisation. The approaches used in research, allow us to trace the dynamics of copper content in fruits of potatoes, tomatoes, cucumbers and onions, during the vegetation periods of 2020-2021 in different soil and climatic zones.Results. It is shown that treatment with copper-containing preparations does not affect the content of copper in fruits of the studied crops during the whole experiment, except for the day of sampling immediately after the treatment. At the same time, reliable excesses in comparison with control samples are observed in experiments, where the aboveground part of crops is investigated. For example, the content of copper in tomato fruits in 2020, taken on the day of the last treatment, was 0.61±0.16 mg/kg and 0.47±0.08 mg/kg in the experimental and control samples, respectively. In 2021, the trend continued and copper concentration on the day of treatment was 0.80±0.15 mg/kg and 0.64±0.12 mg/kg respectively in the experimental and control samples. In the study of onion treated with copper oxychloride-based preparation, the concentration of copper in the whole plant (leaves + bulb) on the day of treatment in 2020 was 8.5±1.1 mg/kg in the experimental sample and 0.43±0.18 mg/kg in the control sample. In 2021, the copper concentration in the experimental sample collected on the treatment day was 14.1±3.8 mg/kg and in the control sample was 1.1±0.2 mg/kg respectively.

Valorization of Seaweed Wracks: Inclusion as Additive in Diets for Grass Carp (Ctenopharyngodon idella).

Macroalgae have been recently described as a potential ingredient for aquafeeds, exerting several physiological benefits. Grass carp (Ctenopharyngodon idella) is a freshwater species, which has been the major fish species produced in the world in the last years. In order to determine the potential use of macroalgal wracks in fish feeding, C. idella juveniles were fed with an extruded commercial diet (CD) or the CD supplemented with 7% of a wind dried-powder (1 mm) from either a multispecific macroalgal wrack (CD + MU7) or a monospecific macroalgal wrack (CD + MO7) obtained from Gran Canaria island (Spain) coasts. After 100 days of feeding, survival, fish weight, and body indexes were determined, and muscle, liver, and digestive tract samples were collected. The total antioxidant capacity of macroalgal wracks was analyzed by assesing the antioxidant defense response and digestive enzymes activity in fish. Finally, muscle proximate composition, lipid classes (LC), and fatty acid (FA) profiles were also studied. Our results suggest that dietary inclusion of macroalgal wracks does not have negative effects on growth, proximate, and lipid composition, antioxidative status, or digestive capacity of C. idella. In fact, both macroalgal wracks caused a general lower fat deposition, and the multispecific wrack enhanced catalase activity in the liver.

A Mass Spectrometry-Based Strategy for Mapping Modification Sites for the Ubiquitin-Like Modifier NEDD8.

The identification of modification sites for ubiquitin and ubiquitin-like modifiers is an essential step in the elucidation of controlled processes. The ubiquitin-like modifier NEDD8 is an important regulator of plethora of biological processes both under homeostatic and proteotoxic stress conditions. Here, we describe a detailed protocol for proteome-wide identification of NEDDylation sites. The approach is based on the use of cell lines stably expressing the NEDD8R74K mutant. Digestion of samples with Lysyl endopeptidase generates peptides with a di-glycine remnant only from proteins modified with NEDD8R74K but not with ubiquitin or ISG15. The isolation of these peptides with anti-di-glycine antibodies (K-ε-GG) allows the identification of NEDDylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS).

QUANTIFICATION OF LEAD CONTAMINATION IN DIFFERENT SAUDI CHOCOLATE BRANDS USING ATOMIC ABSORPTION TECHNIQUE

Introduction: Although chocolate and candies are widely preferred snacks for children and adults, they can carry a risk of lead (Pb) contamination. The lead is considered as a toxic heavy metal that can cause many diseases to adults, including renal, reproductive, nervous, immune, and cardiovascular systems diseases. In children, lead can cause behavioral, developmental diseases and learning deficits. Aim and objectives:Our aim in this study is to measure the lead concentrations in different Saudi chocolate brands from different Saudi cities in order to compare it to the American FDA recommended levels of lead.Secondary objective: to assess if chocolate lead contamination contributes to the high blood lead levels that was found in our pediatric population. Methods: Ten different chocolate brands products were brought from different Saudi cities markets these samples were digested using (MARS 6) Microwave Digestion system. The clear digested samples were diluted to 50 ml and subjected for the trace elements determination by Flame atomic absorption spectrometry using AAS-7000. Results:All chocolate products that we investigated have shown lead levels between (0.00006 to 0.00654 ppm) which are below the recommended levels. Conclusion:Depending on our findings, the different chocolate brands that we have studied are within the acceptable ranges of lead contamination, as recommended by the American FDA. Further studies should focus on other causes of lead exposure as we have a high blood lead levels in our pediatric population.

Development and validation of a LC–MS/MS method for quantitation of recombinant human growth hormone in rat plasma and application to a pharmacokinetic study

Recombinant human growth hormone (rhGH) is a peptide comprising 191 amino acids, that is mainly used to promote the growth of children and plays an important antiaging role. In the present study, a simple and sensitive quantitation method for rhGH in rat plasma was established by LCMS/MS. After simple and rapid enzymatic digestion of the plasma sample, two suitable surrogate peptides (LFDNAMLR and FPTIPLSR) were selected for quantitative analysis. The results showed good linearity over calibration range 10–2000 ng/mL. The quality control (QC) accuracy ranged from −13.8 to 14.3%, and the accuracy of the lower limit of quantification (LLOQ) ranged from −12.9 to 19.0%. The intra-day and inter-day precision ranges for all QCs were 1.7–13.6% and 4.0–7.0%, respectively. The method was successfully applied to intravenous and subcutaneous pharmacokinetic studies in rats. In comparison with previously published methods, our method features simple sample preparation combined with a short sample processing time (3.5 h), wide linear range (10–2000 ng/mL), small plasma volume (35 μL), and LLOQ (10 ng/mL).

Evaluating the in vitro digestion of lipids rich in medium-chain fatty acids (MCFAs) using dynamic and static protocols

Investigating the digestion of lipids is paramount for developing new lipid-based products. This work evaluated the gastrointestinal (GI) digestion of medium-chain fatty acids (MCFAs) rich lipids. The dynamic GI in vitro system was used to simulate gastric, duodenal, jejunal, and ileal GI tract portions. Results from the dynamic protocol were compared against static in vitro assays and GC analyses were conducted to assess the FA profile of FFA released during digestion. Caprylic and capric acids released during the gastric digestion of MCT oil varied from 61–63% and 36–38% of total esterified FA, respectively. Lauric acid was the most representative FFA released (31–54%) during the gastric digestion of coconut oil samples. It was observed that the gastric digestion phase plays a crucial role in the MCFA lipolysis and the lipase activity restricted the amount of free MCFA liberated during the GI digestion, resulting in incomplete lipids hydrolysis.

Fast and Sensitive Determination of Cadmium and Selenium in Rice by Direct Sampling Electrothermal Vaporization Inductively Coupled Plasma Mass Spectrometry.

In this work, a direct solid sampling device based on modified graphite furnace electrothermal vaporization (GF-ETV) with inductively coupled plasma mass spectrometry (ICP-MS) was established for the simultaneous detection of trace selenium and cadmium in rice samples. A bypass gas was first designed in GF-ETV to improve the device’s analytical sensitivity and precision. The ashing and vaporization conditions, the flow rates of the Ar carrier and the bypass gases of ICP-MS were all investigated. Under the optimized conditions, the limits of detection (LODs) for Se and Cd were 0.5 μg kg−1 and 0.16 μg kg−1, respectively; the relative standard deviations (RSDs) of repeated measurements were within 8% (n = 6). The recoveries of Cd and Se in rice samples were in the range of 89−112% compared with the microwave digestion ICP-MS method, indicating good accuracy and precision for the simultaneous detection of Se and Cd in rice matrix. The whole analysis time is <3 min without the sample digestion process, fulfilling the fast detection of Se and Cd in rice samples to protect food safety.

A novel one-pot fluorescence tagging and depyrimidination strategy for quantification of global DNA methylation

DNA methylation is intensively studied in medical science. Current HPLC methods for quantification of global DNA methylation involve digestion of a DNA sample and HPLC determination of both cytosine (C) and 5-methylcytosine (5mC) so that percentage of 5mC in total cytosine can be calculated as DNA methylation level. Herein we report a novel HPLC method based on a one-pot fluorescence tagging and depyrimidination reaction between DNA and chloroacetaldehyde (CAA) for highly sensitive quantification of global DNA methylation. In the one-pot reaction, C and 5mC residues in a DNA sequence react with CAA, forming fluorescent etheno-adducts that are then released from the sequence through depyrimidination. Interestingly, etheno-5mC (ε-5mC) is ∼20 times more fluorescent than ε-C and other ε-nucleobases resulting from the reaction, which greatly facilitates the quantification. Further, due to the tagging-induced increase in structural aromaticity, ε-nucleobases are far more separable by HPLC than intact nucleobases. The proposed HPLC method with fluorescence detection (HPLC-FD) is quick (i.e., < 1h per assay) and highly sensitive with a detection limit of 0.80 nM (or 250 fg on column) for 5mC. Using the method, DNA samples isolated from yeast, HCT-116 cells, and tissues were analyzed. Global DNA methylation was measured to be in the range from 0.35% to 2.23% in the samples analyzed. This sensitive method allowed accurate analyses of minute DNA samples (∼100 ng) isolated from milligrams of tissues.

Comparison of intact protein and digested peptide techniques for high throughput proteotyping of ApoE

IntroductionApolipoprotein E (ApoE) genotyping has been shown to have diagnostic value in the evaluation of cardiovascular diseases and neurodegenerative disorders such as Alzheimer’s disease. Although genetic testing is well established for this application, liquid chromatography-mass spectrometry (LC–MS) has the potential to provide a high throughput, low-cost alternative for ApoE evaluation.MethodsSerum samples were analyzed by peptide, intact protein, and genomic techniques. For peptide analysis, samples were digested with trypsin followed by liquid chromatography-tandem mass spectrometry analysis (LC–MS/MS) using a high-throughput multichannel LC system coupled to a Sciex 7500 mass spectrometer. For intact protein analysis, ApoE was immuno-purified using a monoclonal antibody immobilized on magnetic beads followed by high-resolution LC–MS analysis using an Exploris 480. DNA was extracted and evaluated using Sanger sequencing as a reference method.Results and discussionThe peptide measurement method produced one discrepant result when compared to genomic sequencing (out of 38 sequenced samples), whereas the intact protein analysis followed by deconvolution resulted in two discrepant results and when the intact protein data was processed with chromatographic integration there were three discrepant results. Therefore, the intact protein method proved slightly less accurate, required longer analysis time, and is substantially more costly, while providing only a 30 min improvement in sample preparation time.ConclusionsWith current MS technology clinical laboratories appear to be better served to utilize trypsin digest sample preparation and LC–MS/MS as opposed to high-resolution LC–MS intact protein analysis techniques for evaluation of ApoE proteotype. Peptide analysis methods are capable of producing accurate results with high throughput and minimal cost.