imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation

An integrated sample preparation method, termed “imFASP”, which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50–500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples.     

A study of reproducibility of guanidination-dimethylation labeling and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry for relative proteome quantification

The combination of dimethylation after guanidination (2MEGA) isotope labeling with microbore liquid chromatography (LC)-matrix-assisted laser desorption ionization (MALDI) MS and MS/MS [C. Ji, N. Guo, L. Li, J. Proteome Res. 4 (2005) 2099] has been reported as a promising strategy for abundance ratio-dependent quantitative proteome analysis. A critical step in using this integrated strategy is to set up the abundance ratio threshold of peptide pairs, above which the peptide pairs are used for quantifying and identifying the protein that is considered to be differentially expressed between two different samples. The threshold is determined by technical variation (i.e., the overall abundance ratio variation caused by the experimental process including sample workup, MS analysis and data processing) as well as biological variation (i.e., the abundance ratio variation caused by the biological process including cell growth), which can be defined and assessed by a coefficient of variation (CV). We have designed experiments and measured three different levels of variations, starting with the same membrane protein preparation, the same batch of cells and three batches of cells from the same cell line grown under the same conditions, respectively. It is shown that technical variation from the experimental processes involved in 2MEGA labeling LC-MALDI MS has a CV of <15%. In addition, the measured biological variation from cell growth was much smaller than the measured technical variation. From the studies of the occurrence rate of outliers in the distribution of the abundance ratio data within a comparative dataset of peptide pairs, it is concluded that, to compare the proteome changes between two sets of cultured cells without the use of replicate experiments, a relative abundance ratio of greater than 2X or less than 0.5X (X is the average abundance ratio of the dataset) on peptide pairs can be used as a stringent threshold to quantify and identify differentially expressed proteins with high confidence.     

Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome.     

Improved 2-nitrobenzenesulfenyl method: optimization of the protocol and improved enrichment for labeled peptides

We have developed the NBS (2-nitrobenzenesulfenyl) method, a quantitative proteome analysis method utilizing stable isotope labeling followed by mass spectrometry. The potential of this method was reported previously, and the procedure has now been further optimized. Here, we describe a procedure utilizing urea or guanidine hydrochloride as a protein denaturant, in conjunction with an improved chromatographic enrichment method for the NBS-labeled peptides using a phenyl resin column. By using this new protocol, both sample loss throughout the protocol and the elution of unwanted unlabeled peptides can be minimized, improving the efficiency of the analysis significantly.     

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed.     

High MS-compatibility of silver nitrate-stained protein spots from 2-DE gels using ZipPlates and AnchorChips for successful protein identification

The availability of easy-to-handle, sensitive, and cost-effective protein staining protocols for 2-DE, in conjunction with a high compatibility for subsequent MS analysis, is still a prerequisite for successful proteome research. In this article we describe a quick and easy-to-use methodological protocol based on sensitive, homogeneous, and MS-compatible silver nitrate protein staining, in combination with an in-gel digestion, employing the Millipore 96-well ZipPlate system for peptide preparation. The improved quality and MS compatibility of the generated protein digests, as compared to the otherwise weakly MS-compatible silver nitrate staining, were evaluated on real tissue samples by analyzing 192 Coomassie-stained protein spots against their counterparts from a silver-stained 2-DE gel. Furthermore, the applicability of the experimental setup was evaluated and demonstrated by the analysis of a large-scale MALDI-TOF MS experiment, in which we analyzed an additional ~1000 protein spots from 2-DE gels from mouse liver and mouse brain tissue.     

Monolithic methacrylate packed 96-tips for high throughput bioanalysis

In the pharmaceutical industry the growing number of samples to be analyzed requires high throughput and fully automated analytical techniques. Commonly used sample-preparation methods are solid-phase extraction (SPE), liquid–liquid extraction (LLE) and protein precipitation. In this paper we will discus a new sample-preparation technique based on SPE for high throughput drug extraction developed and used by our group. This new sample-preparation method is based on monolithic methacrylate polymer as packing sorbent for 96-tip robotic device. Using this device a 96-well plate could be handled in 2–4 min. The key aspect of the monolithic phase is that monolithic material can offer both good binding capacity and low back-pressure properties compared to e.g. silica phases. The present paper presents the successful application of monolithic 96-tips and LC–MS/MS by the sample preparation of busulphan, rescovitine, metoprolol, pindolol and local anaesthetics from human plasma samples and cyklophosphamid from mice blood samples.     

Liquid–liquid extraction combined with differential isotope dimethylaminophenacyl labeling for improved metabolomic profiling of organic acids

A large fraction of the known human metabolome belong to organic acids. However, comprehensive profiling of the organic acid sub-metabolome is a major analytical challenge. In this work, we report an improved method for detecting organic acid metabolites. This method is based on the use of liquid–liquid extraction (LLE) to selectively extract the organic acids, followed by using differential isotope p-dimethylaminophenacyl (DmPA) labeling of the acid metabolites. The ¹²C-/¹³C-labeled samples are analyzed by liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC–FTICR–MS). It is shown that this LLE DmPA labeling method offers superior performance over the method of direct DmPA labeling of biofluids such as human urine. LLE of organic acids reduces the interference of amine-containing metabolites that may also react with DmPA. It can also remove water in a biofluid that can reduce the labeling efficiency. Using human urine as an example, it is demonstrated that about 2500 peak pairs or putative metabolites could be detected in a 30-min gradient LC–MS run, which is about 3 times more than that detected in a sample prepared using direct DmPA labeling. About 95% of the 1000 or so matched metabolites to the Human Metabolome Database (HMDB) are organic acids. It is further shown that this method can be used to handle as small as 10 μL of urine. We believe that this method opens the possibility of generating a very comprehensive profile of the organic acid sub-metabolome that will be useful for comparative metabolomics applications for biological studies and disease biomarker discovery.     

Solid-phase microextraction in bioanalysis: New devices and directions

The primary objective of this review is to discuss recent technological developments in the field of solid-phase microextraction that have enhanced the utility of this sample preparation technique in the field of bioanalysis. These developments include introduction of various new biocompatible coating phases suitable for bioanalysis, such as commercial prototype in vivo SPME devices, as well as the development of sampling interfaces that extend the use of this methodology to small animals such as mice. These new devices permit application of in vivo SPME to a variety of analyses, including pharmacokinetics, bioaccumulation and metabolomics studies, with good temporal and spatial resolution. New calibration approaches have also been introduced to facilitate in vivo studies and provide fast and quantitative results without the need to achieve equilibrium. In combination with the drastic improvement in the analytical sensitivity of modern liquid chromatography–tandem mass spectrometry instrumentation, full potential of in vivo SPME as a sample preparation tool in life sciences can finally be explored. From the instrumentation perspective, SPME was successfully automated in 96-well format for the first time. This opens up new opportunities for high-throughput applications (>1000 samples/day) such as for the determination of unbound and total drug concentrations in complex matrices such as whole blood with no need for sample pretreatment, studies of distribution of drugs in various compartments and/or determination of plasma protein binding and other ligand–receptor binding studies, and this review will summarize the progress in this research area to date.     

Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analysis

A new sample preparation method for MALDI based on the use of a mixture of the two commonly used matrices, DHB and CHCA, is described. The matrix mixture preparation results in increased sequence coverage and spot-to-spot reproducibility for peptide mass mapping compared to the use of the single matrix components. This results in more reliable protein identification in proteomics studies and facilitates automated data acquisition. This method shows better tolerance towards salts and impurities, eliminating the need for pre-purification of the samples. It has also been found to be advantageous for the analysis of intact proteins, and especially for glycoproteins. The mixture allows the presence of rather high concentrations of urea in the sample solutions.     

Optimization of protein sample preparation for two-dimensional electrophoresis

Optimized 2DE sample preparation protocols that maximize total protein recovery are fundamental to improving proteome capture and increasing the utility of 2DE, which is in part limited by inadequate recovery of proteins with diverse physicochemical properties. Maintaining protein solubility is an important factor for protein recovery, but the multitude of solubility-enhancing agents and the relatively low-throughput nature of 2DE limit the systematic study of sample preparation. In this work, design of experiment (DOE) approaches are used to optimize protein recovery by altering the levels of four solubility-enhancing agents (urea, DTT, CHAPS, and SDS) in the initial suspension solution. Protein recovery is quantified by a total protein concentration assay, which is demonstrated to be representative of SDS-PAGE and 2DE recovery. DOE methodologies are presented as relatively high-throughput procedures for optimizing 2DE sample preparation parameters for a variety of sample types. Optimal suspension solution compositions are shown to vary across a model protein solution (no urea or DTT), Chinese hamster ovary (CHO) cell lysate (8 M urea, ≥2% CHAPS, ≥32.5 mM DTT), and Escherichia coli cell lysate (8 M urea, 4% CHAPS, 65 mM DTT), with optimized conditions increasing 2DE protein recovery at least 50% compared to suboptimal conditions.     

A Parametric Study of Sample Lysis and DNA Purification Techniques for Use in Automated Devices

Abstract

We present a parametric study on the efficiency of several automatable procedures for the extraction and purification of DNA from a variety of pathogens. Based on the results of this work, an optimized protocol has been developed for use with both spiked buffers and nasal wash. All steps of this protocol are suitable for incorporation into a field-portable, automated sample preparation device. From introduction of the sample to elution of DNA, the entire process was completed in less than 60 min, and the time could be reduced further by automation. The recovered DNA is of sufficient quality for real-time or multiplex PCR amplification. The protocol was demonstrated on nasal wash samples spiked with E. coli containing the J7R and crmB genes. Subsequent testing on resequencing microarrays correctly identified the samples as Variola major virus.     

Extensible automated dispersive liquid–liquid microextraction

In this study, a convenient and extensible automated ionic liquid-based in situ dispersive liquid–liquid microextraction (automated IL-based in situ DLLME) was developed. 1-Octyl-3-methylimidazolium bis[(trifluoromethane)sulfonyl]imide ([C₈MIM]NTf₂) is formed through the reaction between [C₈MIM]Cl and lithium bis[(trifluoromethane)sulfonyl]imide (LiNTf₂) to extract the analytes. Using a fully automatic SPE workstation, special SPE columns packed with nonwoven polypropylene (NWPP) fiber, and a modified operation program, the procedures of the IL-based in situ DLLME, including the collection of a water sample, injection of an ion exchange solvent, phase separation of the emulsified solution, elution of the retained extraction phase, and collection of the eluent into vials, can be performed automatically. The developed approach, coupled with high-performance liquid chromatography–diode array detection (HPLC–DAD), was successfully applied to the detection and concentration determination of benzoylurea (BU) insecticides in water samples. Parameters affecting the extraction performance were investigated and optimized. Under the optimized conditions, the proposed method achieved extraction recoveries of 80% to 89% for water samples. The limits of detection (LODs) of the method were in the range of 0.16–0.45 ng mL⁻¹. The intra-column and inter-column relative standard deviations (RSDs) were <8.6%. Good linearity (r > 0.9986) was obtained over the calibration range from 2 to 500 ng mL⁻¹. The proposed method opens a new avenue for automated DLLME that not only greatly expands the range of viable extractants, especially functional ILs but also enhances its application for various detection methods. Furthermore, multiple samples can be processed simultaneously, which accelerates the sample preparation and allows the examination of a large number of samples.     

Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry

The combination of gel-based two-dimensional protein separations with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the workhorse for the large-scale analyses of proteomes. Such high-throughput proteomic approaches require automation of all post-separation steps and the in-gel digest of proteins especially is often the bottleneck in the protein identification workflow. With the objective of reaching the same high performance of manual low-throughput in-gel digest procedures, we have developed a novel stack-type digestion device and implemented it into a commercially available robotic liquid handling system. This modified system is capable of performing in-gel digest, extraction of proteolytic peptides, and subsequent sample preparation for MALDI-MS without any manual intervention, but with a performance at least identical to manual procedures as indicated on the basis of the sequence coverage obtained by peptide mass fingerprinting. For further refinement of the automated protein identification workflow, we have also developed a motor-operated matrix application device to reproducibly obtain homogenous matrix preparation of high quality. This matrix preparation was found to be suitable for the automated acquisition of both peptide mass fingerprint and fragment ion spectra from the same sample spot, a prerequisite for high confidence protein identifications on the basis of peptide mass and sequence information. Due to the implementation of the stack-type digestion device and the motor-operated matrix application device, the entire platform works in a reliable, cost-effective, and sensitive manner, yielding high confidence protein identifications even for samples in the concentration range of as low as 100 fmol protein per gel plug.      

Expeditious and scalable preparation of a Li-Thiele reagent for amine-based bioconjugation

Chemoselective amine bioco njugation has long been a challenge for native protein modification.Inspired by Thiele's seminal discovery,Li and co-workers recently developed an orto-phthalaldehyde(OPA)based reagent for labeling the amino group of a protein.Here we report an expeditious and scalable synthesis of a Li-Thiele reagent featuring an arene construction strategy.The reagent contains an alkyne side chain as a handle for secondary modification.     

Selective determination of estrogenic compounds in water by microextraction by packed sorbents and a molecularly imprinted polymer coupled with large volume injection-in-port-derivatization gas chromatography-mass spectrometry

A fully automated protocol consisting of microextraction by packed sorbents (MEPS) coupled with large volume injection-in-port-derivatization-gas chromatography–mass spectrometry (LVI-derivatization-GC–MS) was developed to determine endocrine disrupting compounds (EDCs) such as alkylphenols, bisphenol A, and natural and synthetic hormons in river and waste water samples. During method optimization, the extraction parameters as ion strength of the water sample, the MEPS extraction regime, the volume of organic solvent used for the elution/injection step, the type of elution solvents and the selectivity of the sorbents were studied. For optimum in-port-derivatization, 10 μL of the derivatization reagent N,O-bis(trimethylsilyl)triufloroacetamide with 1% of trimethylchlorosilane (BSTFA + 1% TMCS) was used. 17β-Estradiol-molecularly imprinted polymer (MIP) and silica gel (modified with C-18) sorbents were examined for the enrichment of the target analytes from water samples and the obtained results revealed the high selectivity of the MIP material for extraction of substances with estrogen-like structures. Recovery values for most of the analytes ranged from 75 to 109% for the C18 sorbent and from 81 to 103% for the MIP material except for equilin (on C18 with only 57–66% recovery). Precision (n = 4) of the entire analysis protocol ranged between 4% and 22% with both sorbents. Limits of detection (LODs) were at the low ng L⁻¹ level (0.02–87, C18 and 1.3–22, MIP) for the target analytes.     

Incorporation of 18Oxygen into Peptide Mixtures and Analysis with Multi‐Dimensional Chromatography and Mass‐Spectroscopy

Abstract

Enzymatic labeling with ¹⁸oxygen has the potential to become a widely applied method of isotope labeling for differential protein expression analysis by mass spectrometry because it is not amino acid specific and the reagents are cost‐effective and readily available. In this work, we investigate experimental parameters that affect efficient ¹⁸O incorporation with a model bovine serum albumin protein system and then use optimized chemistries for labeling the c‐terminus of peptides in a yeast proteome. Additionally, the role of sample handling, including the use of liquid chromatography was examined. An analytical methodology was developed which demonstrates the application of multi‐dimensional chromatography in conjunction with enzymatic labeling.     

Maximizing recovery of water-soluble proteins through acetone precipitation

Solvent precipitation is commonly used to purify protein samples, as seen with the removal of sodium dodecyl sulfate through acetone precipitation. However, in its current practice, protein loss is believed to be an inevitable consequence of acetone precipitation. We herein provide an in depth characterization of protein recovery through acetone precipitation. In 80% acetone, the precipitation efficiency for six of 10 protein standards was poor (ca. ≤15%). Poor recovery was also observed for proteome extracts, including bacterial and mammalian cells. As shown in this work, increasing the ionic strength of the solution dramatically improves the precipitation efficiency of individual proteins, and proteome mixtures (ca. 80–100% yield). This is obtained by including 1–30 mM NaCl, together with acetone (50–80%) which maximizes protein precipitation efficiency. The amount of salt required to restore the recovery correlates with the amount of protein in the sample, as well as the intrinsic protein charge, and the dielectric strength of the solution. This synergistic approach to protein precipitation in acetone with salt is consistent with a model of ion pairing in organic solvent, and establishes an improved method to recover proteins and proteome mixtures in high yield.     

A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents

A new solid-phase, fluorescence-based protein assay was developed that quantifies proteins in the presence of detergents, urea and reducing agents (one-dimensional sodium dodecyl sulfate (1-D SDS) lysis buffers and urea isoelectric focusing (IEF) buffers). A specially designed 96-well microplate facilitates application of protein samples to the assay paper and allows easy quantitation of samples using fluorescence microplate readers (top or bottom reading format). Alternatively, stained membranes may be directly scanned using a variety of different laser or charge-coupled device (CCD)-based imaging devices with UV or visible imaging capabilities. Since protein is specifically bound to the membrane, contaminants are readily washed away, avoiding interference with the protein measurement. The protein assay has a dynamic range extending from 10 ng to 5 microg of protein per microliter and requires only 1 microL of sample, which is ideal for samples destined for electrophoresis. The protein-to-protein variability of staining of ten different proteins was determined to be comparable with that of the bicinchoninic acid (BCA) and Lowry assays (16%). Additionally, the quality of the assay according to Z-factor analyses is excellent.     

Evaluation of isotope-coded protein labeling (ICPL) in the quantitative analysis of complex proteomes

An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE “classical” approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification.