Sample Processing Methods Research Articles

Tissue Sample Preparation for In Vivo Rodent Alkaline Comet Assay

The Japanese Environmental Mutagen Society/the Mammalian Mutagenicity Study group conducted a collaborative study to investigate whether cell nuclei or whole cells might be more suitably used to correctly detect genotoxic chemicals in the in vivo rodent alkaline Comet assay. Four participating laboratories applied four sample processing methods, i.e., three homogenization methods using the usual Potter-type shaft, a customized (loose) Potter-type shaft, or a Downs-loose-type shaft, for preparing cell nuclei, and the mesh membrane method for preparing whole cells, to the male rat liver. Homogenization with the usual Potter-type shaft clearly produced damage of the cell nuclei and DNA, while the other three methods seemed to provide similar conditions of the tissue samples. The proportion of cell nuclei: whole cells was 80-90%: 10-20% in all laboratories when the samples were prepared by homogenization using a Downs-loose-type shaft or by the mesh membrane method. The %DNA in tail were comparable in both samples among the negative control groups (single oral administration with physiological saline) of all laboratories, and showed an equal degree of increase in both samples of the ethyl methanesulfonate groups (single oral administration at 250 mg/kg) in all laboratories. In conclusion, the homogenization method using a loosely customized Potter-type shaft or a Downs-loose-type shaft, and the mesh membrane method would be equally acceptable for the in vivo rodent alkaline Comet assay.

P49 Intra-subject variation in breath profile of exhaled volatile organic compounds (eVOCs) in chronic obstructive pulmonary disease (COPD)

IntroductionSpecific patterns of exhaled volatile compounds (eVOCs) have been described in a variety of diseases including lung cancer, tuberculosis and COPD, but there are no standardised and universally acceptable methods...

Approach To Evaluating Dried Blood Spot Sample Stability during Drying Process and Discovery of a Treated Card To Maintain Analyte Stability by Rapid On-Card pH Modification

Unstable drug candidates often lead to complexity for both sample collection and bioanalysis. Dried blood spot (DBS) technology is believed to be a viable solution to address this problem. However, it is currently a challenge to evaluate compound stability on DBS due to its solid format. The observed compound loss on a DBS card could be degradation and/or incomplete recovery. Therefore, a reliable bioanalytical method which can differentiate recovery loss from degradation is necessary for such stability evaluation. In this paper, the stability of an unstable drug candidate (KAI-9803) in human blood was evaluated using DBS. A reliable approach to evaluating analyte stability on DBS was developed with an appropriate time-zero sample, a consistent DBS sample processing method, and a suitable positive control. Commercially available DBS cards were evaluated, and it was found that KAI-9803 degraded during the drying process. An in-house modified DBS card was developed and demonstrated to be able to stabilize KAI-9803 during the drying process by rapidly lowering the pH of the spotted blood sample. The storage stability of KAI-9803 in human blood on this new card has been established for at least 48 days at room temperature. This in-house modified DBS card could provide a generic approach for other compounds which require stabilization at a low pH.

Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield

ObjectiveCell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used.MethodsMicrofluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples.ResultsTotal cell-free DNA quantity increased significantly with time in samples stored in K3EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4°C did not prevent these changes.ConclusionWhen samples can be processed within eight hours of blood draw, K3EDTA tubes can be used. Prolonged transfer times in K3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.

Sample collection and handling considerations for peptidomic studies in whole saliva; implications for biomarker discovery

BackgroundProteomic studies in saliva have demonstrated its potential as a diagnostic biofluid, however the salivary peptidome is less studied. Here we study the effects of several sample collection and handling factors on salivary peptide abundance levels. MethodsSalivary peptides were isolated using an ultrafiltration device and analyzed by tandem mass spectrometry. A panel of 41 peptides common after various treatments were quantified and normalized. We evaluated the effects of freezing rate of the samples, nutritional status of the donors (fed vs. fasted), and room-temperature sample degradation on peptide abundance levels. Repeatability of our sample processing method and our instrumental analysis method were investigated. ResultsIncreased sample freezing rate produced higher levels of peptides. Donor nutritional status had no influence on the levels of measured peptides. No significant difference was detected in donors' saliva following 5, 10 and 15min of room-temperature degradation. Sample processing and instrumental variability were relatively small, with median CVs of 9.6 and 6.6. ConclusionsPeptide abundance levels in saliva are rather forgiving towards variations in sample handling and donor nutritional status. Differences in freezing methods may affect peptide abundance, so consistency in freezing samples is preferred. Our results are valuable for standardizing sample collection and handling methods for peptidomic-based biomarker studies in saliva.

Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

Validation of Rapid Methods for Enumeration of Markers for Human Sewage Contamination in Recreational Waters

Rapid methods of water quality assessment are essential for informing the public and officials of sewage-polluted water in a timely manner. Since fecal indicator bacteria can be contributed to the water by many sources, not all of which have equal risk for human health, rapid, quantitative methods that can discriminate human from non-human fecal contamination are informative for risk assessment and to identify problems in infrastructure or land use that contribute to degradation of water quality. This work compared standard, membrane filtration-based measures of the fecal indicator bacteria E. coli and enterococci to qPCR methods in terms of quantitative correlation and consistency of performance in various types of waters. Furthermore, the genes of two human-associated microorganisms, one from the bacterial group Bacteroidales and one from human polyomaviruses JCV and BKV, were used to quantitatively assess the presence of human sewage using qPCR in various types of subtropical waters in Florida, including fresh, brackish and salt waters. The qPCR methods performed well across the range of water types, although highly tannic water types (e.g. swamp-fed river) were more likely to be subject to inhibition than lake, estuarine or marine waters. Clean-up of inhibited samples by chemical methods produced highly variable results, therefore dilution of template material proved to be a better option for relieving inhibition. Concentrations of qPCR targets for which culture-dependent methods exist were generally in agreement when culture and qPCR methods were compared. Comparison of three different sample processing methods showed that the EPA CE and PE methods were superior to the USF methods for bacterial targets, but not for HPyVs. The EPA CE method had less sample-sample variability than the PE method, but also had a higher detection limit, making it potentially less sensitive than the PE method. This title belongs to WERF Research Report Series . ISBN: 9781780400020 (eBook)

Combining Laser Ablation/Liquid Phase Collection Surface Sampling and High-Performance Liquid Chromatography−Electrospray Ionization-Mass Spectrometry

This letter describes the coupling of ambient pressure transmission geometry laser ablation with a liquid phase sample collection method for surface sampling and ionization with subsequent mass spectral analysis. A commercially available autosampler was adapted to produce a liquid droplet at the end of the syringe injection needle while in close proximity to the surface to collect the sample plume produced by laser ablation. The sample collection was followed by either flow injection or a high-performance liquid chromatography (HPLC) separation of the extracted components and detection with electrospray ionization mass spectrometry (ESI-MS). To illustrate the analytical utility of this coupling, thin films of a commercial ink sample containing rhodamine 6G and of mixed isobaric rhodamine B and 6G dyes on glass microscope slides were analyzed. The flow injection and HPLC-ESI-MS analysis revealed successful laser ablation, capture, and with HPLC, the separation of the two compounds. The ablated circular area was about 70 μm in diameter for these experiments. The spatial sampling resolution afforded by the laser ablation, as well as the ability to use sample processing methods like HPLC between the sample collection and ionization steps, makes this combined surface sampling/ionization technique a highly versatile analytical tool.

Development of optimal liquid based cytology sample processing methods for HPV testing: Minimising the ‘inadequate’ test result

Incorporation of HPV testing into cervical screening is anticipated and robust methods for DNA extraction from liquid based cytology (LBC) samples are required. This study compared QIAamp extraction with Proteinase K digestion and developed methods to address DNA extraction failure (β-globin PCR negative) from clinical specimens.Proteinase K and QIAamp extraction methods in paired LBC samples were comparable with adequate DNA retrieved from 93.3% of clinical specimens. An HPV prevalence cohort (n=10,000) found 7% (n=676) LBC samples tested negative for β-globin, and were classified as inadequate. This ‘failure’ rate is unsuitable for population screening, particularly as the sampling method is intrusive. 379/676 samples were assessed to determine the cause of test failure. Re-testing confirmed adequate DNA in 21.6% of the original extracts; re-extraction from stored material identified 56.2% samples contained adequate material; dilution to overcome sample inhibition (1:10) resolved 51.7% cases in original extracts and 28% in new extracts.A standardised approach to HPV testing with an optimal DNA concentration input rather than standard volume input is recommended. Samples failing initial DNA extraction should be repeat extracted and assessed for sample inhibition to reduce the 7% of HPV tests being reported as inadequate and reduce the need for retesting of those women to <1%.

Systematic evaluation of pre-HPLC sample processing methods on total and individual isoflavones in soybeans and soy products

There are mainly two protocols in isoflavone analysis, one that involves hydrolysis prior to HPLC analysis and the other direct HPLC analysis. In this study, three different hydrolysis methods were systematically re-evaluated, and compared with direct HPLC analysis. Acidic hydrolysis (1.2–3 M HCl in ethanol at 80 °C) showed a maximum conversion of ca. 92% from glucosides to aglycones in 2 h; however, longer reaction caused degradation of genistein. Alkaline hydrolysis using 2 M NaOH converted acetylglucosides and malonylglucosides to their respective glucosides within 10 min. Glucuronidase from H. pomatia effectively converted isoflavone glucosides and acetylglucosides to aglycones. Quantification of the total isoflavones in various soy food products showed no significant difference among direct injection and the three hydrolysis methods ( P < 0.05). We conclude that direct analysis of isoflavones in crude extracts is a rapid and accurate method to obtain isoflavone profiles and compositions in soybean, soy foods and beverages.

Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity

Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.

A Protein Processing Filter Method for Bacterial Identification by Mass Spectrometry-Based Proteomics

A "one-pot" alternative method for processing proteins and isolating peptide mixtures from bacterial samples is presented for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and data reduction. The conventional in-solution digestion of the protein contents of bacteria is compared to a small disposable filter unit placed inside a centrifuge vial for processing and digestion of bacterial proteins. Each processing stage allows filtration of excess reactants and unwanted byproduct while retaining the proteins. Upon addition of trypsin, the peptide mixture solution is passed through the filter while retaining the trypsin enzyme. The peptide mixture is then analyzed by LC-MS/MS with an in-house BACid algorithm for a comparison of the experimental unique peptides to a constructed proteome database of bacterial genus, specie, and strain entries. The concentration of bacteria was varied from 10 × 10(7) to 3.3 × 10(3) cfu/mL for analysis of the effect of concentration on the ability of the sample processing, LC-MS/MS, and data analysis methods to identify bacteria. The protein processing method and dilution procedure result in reliable identification of pure suspensions and mixtures at high and low bacterial concentrations.

Rapid and Direct Detection of Apolipoprotein E Genotypes Using Whole Blood from Humans

Polymerase chain reaction (PCR) is a powerful molecular biological tool in the field of toxicity testing and diagnostics. The use of PCR for large-scale genetic testing requires an effective method of sample processing. Unfortunately, isolation of PCR-quality DNA is time-consuming. PCR performed directly on whole blood is preferred because of time efficiency, cost of the procedure, and possible automation for large-scale toxicity evaluation and diagnosis. The apolipoprotein E (APOE) gene contains two single-nucleotide polymorphisms (SNP) located at codons 112 and 158, producing three APOE protein isoforms known to be associated with the risks of developing cardiovascular disease and susceptibility to Alzheimer's disease. In the present study, an attempt was made to use the AnyDirect solution for APOE genotyping by PCR using whole blood directly without DNA purification. Results for two PCR methods, (1) conventional PCR using purified DNA and conventional buffer and (2) direct PCR using whole blood and AnyDirect solution, were compared in four different PCR-based APOE genotyping methods including PCR restriction-fragment-length polymorphism (PCR-RFLP), allele-specific PCR, SNaPshot mini-sequencing, and multiplex tetra-primer amplification refractory mutation system (T-ARMS) PCR. There was complete concordance in the APOE genotypes between conventional PCR and direct PCR, in all four different PCR-based APOE genotyping methods. Data demonstrated that the four different PCR-based APOE genotyping methods are able to determine the APOE genotypes successfully using whole blood directly with the use of AnyDirect solution. The direct multiplex T-ARMS PCR using whole blood may be the most rapid, simple, and inexpensive method for detecting APOE genotypes among four different APOE genotyping methods.

A Generalized Cauchy Distribution Framework for Problems Requiring Robust Behavior

Statistical modeling is at the heart of many engineering problems. The importance of statistical modeling emanates not only from the desire to accurately characterize stochastic events, but also from the fact that distributions are the central models utilized to derive sample processing theories and methods. The generalized Cauchy distribution (GCD) family has a closed-form pdf expression across the whole family as well as algebraic tails, whichmakes it suitable formodeling many real-life impulsive processes. This paper develops a GCD theory-based approach that allows challenging problems to be formulated in a robust fashion. Notably, the proposed framework subsumes generalized Gaussian distribution (GGD) family-based developments, thereby guaranteeing performance improvements over traditional GCD-based problem formulation techniques. This robust framework can be adapted to a variety of applications in signal processing. As examples, we formulate four practical applications under this framework: (1) filtering for power line communications, (2) estimation in sensor networks with noisy channels, (3) reconstruction methods for compressed sensing, and (4) fuzzy clustering.

Critical evaluation of solid waste sample processing for DNA-based microbial community analysis

Landfills represent a unique microbial ecosystem and play a significant role in global biogeochemical processes. The study of complex ecosystems such as landfills using DNA-based techniques can be advantageous since they allow for analysis of uncultured organisms and offer higher resolution in measuring demographic and metabolic (functional) diversity. However, sample acquisition and processing from refuse is challenging due to material heterogeneity. Decomposed refuse was used to evaluate the effect of seven sample processing methods on Bacteria and Archaea community structure using T-RFLP. Bias was assessed using measured richness and by comparing community structure using multi-dimensional scaling (MDS). Generally, direct methods were found to be most biased while indirect methods (i.e., removal of cellular material from the refuse matrix before DNA extraction) were least biased. An indirect method using PO₄ buffer gave consistently high bacterial and archaeal richness and also resulted in 28 and 34% recovery of R. albus and M. formicicum spiked into refuse, respectively. However, the highest recovery of less abundant T-RFs was achieved using multiple processing methods. Results indicate differences in measured T-RF diversity from studies of landfill ecosystems could be caused by methodological (i.e., processing method) variation rather than refuse heterogeneity or true divergence in community structure.

Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing

To detect the site of mutation in RRDR of rpo B gene for rifampicin resistance in MDR-TB by DNA sequencing. 50 MDR-TB patients were enrolled in our study after informed written consent. Mycobacterial DNA was extracted from sputum samples by Universal Sample Processing (USP) method and RRDR of rpo B gene was amplified by PCR using primers RP4T and RP8T and then sequenced by automated DNA sequencing. The nucleotide sequences of RRDR of rpo B gene were compared with the reference sequence. We observed three different types of mutation in the RRDR of rpo B gene. The frequency of mutation in codon 531 (TCG→TTG), 526 (CAC→TAC) and 516 (GAC→GTC) are 60, 26.6 and 6.6% respectively. Of the total cases studied, 6.6% cases, although resistant to rifampicin, did not show any mutation in the RRDR of rpo B gene. Codon 531 (TCG→TTG) is the most common site of mutation in RRDR of rpo B gene for rifampicin resistance in MDR-pulmonary tuberculosis followed by codon 526 (CAC→TAC) and codon 516 (GAC→GTC). The online version of this article (doi:10.1007/s12291-010-0065-3) contains supplementary material, which is available to authorized users.

Influence of mesh size and core penetration on estimates of deep-sea nematode abundance, biomass, and diversity

It is essential to maximize the information that can be gathered in deep-sea studies by thoroughly assessing sample processing methods. Nematodes are commonly used for the study and monitoring of deep-sea floor habitats, but the potential effects of different methods on the quantification of community attributes remain to be quantified. Here, we consider key methodological elements by comparing the effect of sediment depth and mesh size (63, 45, and 32 μm) on: (1) estimates of nematode community attributes, and (2) the sampling effort required to detect changes in these attributes at a bathyal site on the Chatham Rise, south-west Pacific Ocean. The 63 μm mesh retained most (95%) of the nematode biomass but a lower proportion (53–71%) of the nematode abundance. Retention efficiency of common species on this mesh ranged from 12 ( Hapalomus sp.) to >88% ( Comesomatidae spp.). The 63 μm mesh yielded significantly lower diversity estimates than the finer meshes, and failed to detect differences in community structure observed using the 45 and 32 μm mesh sizes. Sediment depth had a substantial effect on all measured community attributes, highlighting the importance of sufficient core penetration into the sediment (≥5 cm) for adequately characterizing nematode distribution. Power analysis showed that using a 32 μm mesh and deepest core penetration led to relatively few (3–8) samples being required to detect significant changes in nematode diversity indices relative to coarser mesh sizes. Characterization of nematode diversity and community structure using appropriate and robust methods of sampling is suggested as a sensitive and efficient tool for the assessment of anthropogenic impacts on deep-sea ecosystems.

PWE-049 Shotgun proteomics in the discovery of biomarkers of non-alcoholic fatty liver disease: an improved methodology

<h3>Introduction</h3> Non-alcoholic fatty liver disease is a common condition affecting up to one third of the US population. The current gold standard diagnostic tool is liver biopsy, which has associated morbidity and mortality concerns, as well as sampling error and inter-observer variability problems. There is an urgent need for the discovery and validation of non-invasive markers of this disease. Many studies have taken a targeted approach, examining the utility of various biochemical and clinical markers thought to be important in the pathogenesis of this condition. Shotgun proteomics offers an untargeted tool for the discovery of protein markers in complex clinical samples. <h3>Methods</h3> Materials: 30 patients with biopsy proven NAFLD were recruited. Patients had negative viral hepatitis screens, negative autoimmune screens, normal iron stores, and were taking no hepatotoxic medications. Biochemical and anthropological measurements were also recorded. <h3>Method</h3> Serum samples were pooled into three groups (SS/NASH/NASH fibrosis, <i>F</i>_1,2, with n=7 per group). The pooled samples were immunodepleted of the top 91 most abundant proteins. Buffer exchange and lyophilisation was performed before samples were reduced alkylated then digested with trypsin. The resultant peptide mixtures were labelled with iTRAQ tags as per the manufacturers9 protocol (Applied Biosystems), and then pooled together. Sample fraction was performed using a reverse phase C18 column at high pH. A total of 88 fractions were collected and combined to 47 fractions based on the appearance of the chromatogram. The 47 fractions were analysed by LC-MS/MS (QTOF, Agilent), using an optimised LC and MS methodology. The resultant MS data were processed using MASCOT Distiller software and the IPI_human database searched for matches. Only proteins with 2 peptide identification and a MASCOT score indicative of identity were included for analysis. <h3>Results</h3> A total of 557 proteins were identified and included for analysis. Previous iTRAQ technical replicate data indicated an error range of 1.13 (±0.10)–1.37 (±0.12). Proteins with fold changes out with this range were considered significantly differentially expressed. At least 10 proteins had a fold change &gt;5 in the NASH early fibrosis group. Interestingly, several protein markers of NAFLD fibrosis previously reported in the literature were also found to be appropriately unregulated in this experiment, including α-2-macroglobulin. <h3>Conclusion</h3> Discussion iTRAQ technology is a powerful tool, allowing analysis of up to 8 different samples simultaneously. We have improved our method of sample preparation and processing to increase the number of protein identifications. The findings from this study require further validation.

Alcohol Fixation of Induced Sputum Samples for Applications in Rural Communities

Sputum induction is a tool recommended for the assessment of airway inflammation and disease management. Currently, its use is limited because samples need to be processed within 3 h of induction (ie, while cells are viable); therefore, this procedure is unavailable to most clinicians. To develop a fixation method for induced sputum samples that allows for a delay in processing while maintaining sample integrity and not altering the standard processing method. Sputum samples were collected and split into three portions: a fresh sample processed using the routine method (within 3 h, using dithiothreitol); fixation in alcohol followed by delayed processing using the routine method (within 48 h to 72 h, using dithiothreitol); and fixation in formaldehyde followed by delayed processing using an alternative method (within 48 h to 72 h, using proteolysis). For each method, cytospins were prepared and differential cell counts were performed. Fixation in alcohol provides accurate measures of eosinophils and macrophages, but not neutrophils. Formaldehyde fixation provides accurate measures of neutrophils and macrophages, but not eosinophils. Alcohol fixation is a superior method for eosinophil quantification. It requires alteration of standardized methods for sputum sample processing and should be recommended for monitoring eosinophilic airway disease in settings where immediate processing of a sputum sample is not possible.

Rapid microscale in-gel processing and digestion of proteins using surface acoustic waves

A new method for in-gel sample processing and tryptic digestion of proteins is described. Sample preparation, rehydration, in situ digestion and peptide extraction from gel slices are dramatically accelerated by treating the gel slice with surface acoustic waves (SAWs). Only 30 minutes total workflow time is required for this new method to produce base peak chromatograms (BPCs) of similar coverage and intensity to those observed for traditional processing and overnight digestion. Simple set up, good reproducibility, excellent peptide recoveries, rapid turnover of samples and high confidence protein identifications put this technology at the fore-front of the next generation of proteomics sample processing tools.