Improvement of a sample preparation method assisted by sodium deoxycholate for mass‐spectrometry‐based shotgun membrane proteomics†

In current shotgun‐proteomics‐based biological discovery, the identification of membrane proteins is a challenge. This is especially true for integral membrane proteins due to their highly hydrophobic nature and low abundance. Thus, much effort has been directed at sample preparation strategies such as use of detergents, chaotropes, and organic solvents. We previously described a sample preparation method for shotgun membrane proteomics, the sodium deoxycholate assisted method, which cleverly circumvents many of the challenges associated with traditional sample preparation methods. However, the method is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of sodium deoxycholate when it is used at relatively low concentrations such as 1%. Hence, we present an enhanced sodium deoxycholate sample preparation strategy that first uses a high concentration of sodium deoxycholate (5%) to lyse membranes and extract/solubilize hydrophobic membrane proteins, and then dilutes the detergent to 1% for a more efficient digestion. We then applied the improved method to shotgun analysis of proteins from rat liver membrane enriched fraction. Compared with other representative sample preparation strategies including our previous sodium deoxycholate assisted method, the enhanced sodium deoxycholate method exhibited superior sensitivity, coverage, and reliability for the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.     

Evaluation of the combinative application of SDS and sodium deoxycholate to the LC–MS‐based shotgun analysis of membrane proteomes

SDS and sodium deoxycholate (SDC) as two representative detergents have been widely used in LC–MS/MS‐based shotgun analysis of membrane proteomes. However, some inherent disadvantages limit their applications such as interference with MS analysis or their weak ability to disrupt membranes. To address this, the combinative application of SDS and SDC was developed and evaluated in our study, which comprehensively used the strong ability of SDS to lyse membranes and solubilize hydrophobic membrane proteins, and the high efficiencies of an optimized acetone precipitation method and SDC in sample clean‐up, protein recovery, and redissolution and digestion of precipitated proteins. The comparative study using a rat‐liver‐membrane‐enriched sample showed that, compared with other three commonly used methods including the filter‐aided sample preparation strategy, the combinative method not only increased the identified number of total proteins, membrane proteins, and integral membrane proteins by an average of 19.8, 23.9, and 24.8%, respectively, but also led to the identification of the highest number of matching peptides. All these results demonstrate that the method yielded better recovery and reliability in the identification of the proteins especially highly hydrophobic integral membrane proteins than the other three methods, and thereby has more potential in shotgun membrane proteomics.     

Methods for generating protein molecular ions in ToF-SIMS

One of the greatest challenges in mass spectrometry lies in the generation and detection of molecular ions that can be used to directly identify the protein from the molecular weight of the molecular ion. Typically, proteins are large (MW > 1000), nonvolatile, and/or thermally labile, but the vaporization process produced by many mass spectrometry techniques including time-of-flight secondary ion mass spectrometry (ToF-SIMS) is inherently limited to generating ions from smaller compounds or fragments of the parent molecule, making the identification of proteins complex. The application of specific molecules to aid in the generation of high molecular weight ions in ToF-SIMS has been recognized for some time. In this study we have developed a matrix-SAM substrate preparation technique based on the self-assembly of a matrix-like molecule, mercaptonicotinic acid (MNA), on gold. We then compare this substrate with two existing ToF-SIMS sample preparation techniques, cationized alkane thiol and matrix-enhanced SIMS (MESIMS). The results of this study illustrate that while there is a range of methods that can be used to improve the molecular ion yield of proteins in ToF-SIMS, their efficacy and reproducibility vary considerably and crucially are linked to the sample preparation and/or protein application methods used. Critically, the MNA modified substrate was able to simultaneously induce molecular ions for each protein present in a multicomponent solution, suggesting that this sample preparation technique may have future application in proteomics and DNA analysis.     

A novel approach for protein identification from complex cell proteome using modified peptide mass fingerprinting algorithm

A unique peptide based search algorithm for identification of protein mixture using PMF is proposed. The proposed search algorithm utilizes binary search and heapsort programs to generate frequency chart depicting the unique peptides corresponding to all proteins in a proteome. The use of binary search program significantly reduces the time for frequency chart preparation to ～2 s for a proteome comprising ～23 000 proteins. The algorithm was applied to a three‐protein mixture identification, host cell protein (HCP) analysis, and a simulation‐generated data set. It was found that the algorithm could identify at least one unique peptide of a protein even in the presence of fourfold higher concentration of another protein. In addition, two HCPs that are known to be difficult to remove were missed by MS/MS approach and were exclusively identified using the presented algorithm. Thus, the proposed algorithm when used along with standard proteomic approaches present avenues for enhanced protein identification efficiency, particularly for applications such as HCP analysis in biopharmaceutical research, where identification of low‐abundance proteins are generally not achieved due to dynamic range limitations between the target product and HCPs.     

The effect of sodium dodecyl sulfate and anion‐exchange silica gel on matrix‐assisted laser desorption/ionization mass spectrometric analysis of proteins

Sodium dodecyl sulfate (SDS), an anionic surfactant, is widely used in peptide and protein sample preparation. When the sample is analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), this surfactant can often cause signal suppression. We have previously reported an on‐probe sample preparation method using a suspension of anion‐exchange silica gel and sinapinic acid (i.e., gel‐SA suspension) as a matrix, thereby greatly improving the MALDI signal detection of the protein solutions containing SDS. In this study, we found that a certain amount of SDS enhanced the MALDI signal intensity for protein samples. This effect was also observed when using sodium decyl sulfate and sodium tetradecyl sulfate instead of SDS. Furthermore, this on‐probe sample preparation method using both SDS and the gel‐SA suspension improved the detection limit of protein samples in the MALDI‐MS analysis by about ten‐fold as compared to that of protein samples without SDS and the gel‐SA suspension. This method can be applied not only to the MALDI‐MS analysis of samples containing SDS, but also to the examination of proteins at femtomole levels or insoluble proteins such as membrane proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samples

Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 microm id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified proteins.     

Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.     

A Micro-Scale Model to Monitor the Major Proteins in Human Urine Before and After Medication with Angiotensin-Converting Enzyme Inhibitor: A Preliminary Study

A simple and micro-scale liquid chromatographic (LC) method coupled with mass spectrometry was developed for analyzing major proteins in human urine. After one-step sample preparation, proteins were precipitated, redissolved, and digested. Only micro-liter level (10 μL) of urine was sufficient for major protein identification. This method was applied in clinical study, and urine proteins were monitored after medication with angiotensin-converting enzyme inhibitor within 24 hr. This method can identify many important proteins in human urine. Bioactive peptides associated with blood pressure control can also be identified simultaneously. We hope this simple method may prove useful in clinical research and disease diagnosis.     

Use of monolithic supports in proteomics technology

An overview on the utilization of monoliths in proteomics technology will be given. Both silica- and polymer-based monoliths have broad use for microseparation of tryptic peptides in reversed-phase (RP) mode before identification by mass spectrometry (MS) or by MS/MS. For two-dimensional (2D) LC separation of peptides before MS or MS/MS analysis, a combination of ion-exchange, usually cation-exchange (CEX) chromatography with RP chromatography on monolithic supports can be employed. Immobilized metal ion affinity chromatography monoliths with immobilized Fe3+-ions are used for the isolation of phosphopeptides. Monoliths with immobilized affinity ligands are usually applied to the rapid separation of proteins and peptides. Miniaturized reactors with immobilized proteolytic enzymes are utilized for rapid on- or offline digestion of isolated proteins or protein mixtures prior to identification by LC-MS/MS. Monoliths also have broad potential for application in sample preparation, prior to further proteomic analyses. Monolithic supports with large pore sizes can be exploited for the isolation of nanoparticles, such as cells, organelles, viruses and protein aggregates. The potential for further adoption of monolithic supports in protein separation and enrichment of low abundance proteins prior to proteolytic digestion and final LC-MS/MS protein identification will be discussed.     

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

The analysis of proteins in biological samples is highly desirable, given their connection to myriad biological functions and disease states, as well as the growing interest in the development of protein‐based pharmaceuticals. The introduction and maturation of “soft” ionization methods, such as electrospray ionization and matrix‐assisted laser desorption/ionization, have made mass spectrometry an indispensable tool for the analysis of proteins. Despite the availability of powerful instrumentation, sample preparation and fractionation remain among the most challenging aspects of protein analysis. This review summarizes these challenges and provides an overview of the state‐of‐the‐art in sample preparation and fractionation of proteins for mass spectrometric analysis, with an emphasis on those used for top‐down proteomic approaches. Biological fluids, particularly important for clinical and pharmaceutical applications and their characteristics are also discussed. While immunoaffinity‐based methods are addressed, more attention is given to non‐immunoaffinity‐based methods, such as precipitation, coacervation, size exclusion, dialysis, solid‐phase extraction, and electrophoresis. These techniques are presented in the context of a significant number of studies where they have been developed and utilized.     

Liquid chromatography–mass spectrometry methods for urinary biomarker detection in metabonomic studies with application to nutritional studies

The effects of sample preparation and chromatographic method differences on the classification and recovery of metabolic biomarkers from UPLC‐MS measurements on urine samples of humans exposed to different dietary interventions have been investigated. Eight volunteers consumed three high‐fat meals (rich in saturated, monounsaturated and polyunsaturated fatty acids, respectively) in randomized order with a washout period in between. For each participant, urine samples were obtained prior to and at three timed intervals after each meal. Samples were processed either by dilution (1 : 4) or by liquid–liquid extraction and then run under two different gradient conditions. For each analysis method, a total of 96 observations (eight participants, four time points, three diets) were measured. The total ion count chromatograms were analyzed using partial‐least‐squares discriminant analysis. All three dietary classes could be discriminated irrespective of sample preparation and chromatographic method. However, the main discriminating metabolites varied according to sample preparation, indicating that sample treatment and chromatographic conditions influence the ability to extract biomolecular information. Diluted samples showed higher m/z compounds (ca 400 u) while liquid–liquid extraction samples showed low m/z at the same retention time span. Optimized methods for metabolite identification (e.g. organic acids) were statistically inferior to global screening for mixed compound identification, confirming that multiple compound class‐based metabolic profiles are likely to give superior metabonomic (diagnostic) classification, although great care has to be taken in the interpretation in relation to matrix effects. Copyright © 2009 John Wiley & Sons, Ltd.     

Conductive carbon tape as a sample platform for microwave-based MALDI MS detection of proteins and phosphoproteins

In this study, we developed a novel microwave-assisted protein preparation and digestion method for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry analysis and identification of proteins that involves using conductive carbon tape as a sample platform for sample preparation (reduction and alkylation) and digestion under microwave heating and as a plate for MALDI analysis. This method allows for the enzymatic digestion products of proteins to be directly analyzed by MALDI mass spectrometry and results in a marked reduction in sample loss. Our protocol requires only a small volume (1 μL) of reaction solvent, which increases the frequency of enzyme-to-protein contact, thereby resulting in more efficient digestion of sample than conventional in-solution digestion methods. To test this protocol, we used magnetic iron (II, III) oxide nanoparticles as concentrating probes to enrich phosphopeptides from a mixture of peptides in enzymatically digested protein samples. We found that the one-pot on-tape-based protein preparation and digestion under microwave heating combined with the on-tape-based enrichment method not only dramatically reduced the time required for phosphopeptides analysis but also allowed for the simultaneous identification of phosphoproteins. The advantages of our protocol include ease of use, high digestion efficiency, high specificity, and rapid (15 min) identification of proteins and enrichment of phosphopeptides in a mixture of enzymatically digested protein samples.     

Improvements in protein identification confidence and proteome coverage for human liver proteome study by coupling a parallel mass spectrometry/mass spectrometry analysis with multi-dimensional chromatography separation

Recently, matrix-assisted laser desorption ionization (MALDI) technique has been shown to be complementary to electrospray ionization (ESI) with respect to the population of peptides and proteins that can be detected. In this study, we tried to hyphenate MALDI-TOF-TOF-MS and ESI-QUADRUPOLE-TOF-MS with a single 2D liquid chromatography for complicated protein sample analysis. The effluents of RPLC were split into two parts for the parallel MS/MS detection. After optimizing the operation conditions in LC separation and MS identification, a total of 1149 proteins were identified from the global lysate of normal human liver (NHL) tissue. Compared to the single MS/MS detection, the combined analysis increased the number of proteins identified (more than 25%) and enhanced the protein identification confidence. Proteins identified were categorized and analyzed based upon their cellular location, biological process and molecular function. The identification results demonstrated the application potential of a parallel MS/MS analysis coupled with multi-dimensional LC separation for complicated protein sample identification, especially for proteome analysis, such as human tissues or cells extracts.     

Recommended criteria for the mass spectrometric identification of target peptides and proteins (<8 kDa) in sports drug testing

Mass spectrometry has become an invaluable tool for the identification of prohibited peptide hormones and proteins in doping control analysis. Regulatory authorities have established criteria for identifying banned drugs in doping control specimens, but these criteria do not address the specific issues for high molecular weight protein drugs such as molecular weight determination of multiply charged molecules, analysis of chemically or enzymatically derived degradation products, identification of amino acid sequence tags, etc. Technical considerations such as sample preparation methods (e.g. immunoaffinity purification), resulting analytes (e.g. intact compounds vs. chemically or enzymatically derived peptides), ionization modes, analyzer resolution, and the information provided by respective techniques are discussed in light of sports drug testing requirements using typical application examples.     

A procedure for the extraction and high resolution two-dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei.

Methods are described for the extraction and preparation of total nuclear proteins for high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post-translational modifications. Unlike other procedures for 2-D PAGE analysis of nuclear proteins the technique allows solubilization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi-labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2-D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2-D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.     

Development of a dedicated peptide tandem mass spectral library for conservation science

In recent years, the use of liquid chromatography tandem mass spectrometry (LC–MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art.     

Acid hydrolysis of proteins in matrix assisted laser desorption ionization matrices

Sample preparation is crucial to the success of experiments in biological mass spectrometry. In proteomics, digestion of the proteins into peptides is a key step for “bottom-up” approaches. Often, the use of enzymes requires physiological conditions, producing peptides that must be extracted or further purified before mass analysis. Chemical cleavage reagents offer more flexibility and can be more compatible with downstream mass analysis. Expanding on prior work using acid hydrolysis, proteolysis with matrix-assisted laser desorption ionization (MALDI) matrices is presented. This sample preparation can be performed rapidly with a minimum of reagents and sample handling, but it must first be evaluated in terms of digestion efficiency, missed cleavages, and side reactions before implementation for in-gel digestion and in-solution digestion using minimal volumes of protein. Time courses of acid hydrolysis are shown for protein standards, illustrating the sensitivity of this type of sample preparation, minimization of side reactions, and performance for proteins in mixtures. To illustrate the potential for sensitive detection of a specific protein, MALDI matrix hydrolysis is used to digest a protein immunoprecipitated from cell lysate.     

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.      

Proteomic profiling of human urine using multi-dimensional protein identification technology

Human urine samples are ideal for proteomic profiling and have tremendous potential as sources of biomarkers. Multi-dimensional protein identification technology (MudPIT) is an effective approach to analyzing human urine or other fluids dominated by diverse metabolites. MudPIT analysis was used to identify 87 proteins in just 15 ml of human urine. A high throughput, reproducible, and sensitive technology, MudPIT may soon be used for more proteomic analyses of metabolites.     

Electrode‐assisted desorption electrospray ionization mass spectrometry

A new ion source has been developed for rapid, noncontact analysis of materials at ambient conditions. The method provides desorption of analytes under ambient conditions directly from different surfaces with little or no sample preparation. The new method, termed electrode‐assisted desorption electrospray ionization (EADESI), is on the basis of the ionization of molecules on different surfaces by highly charged droplets produced on a sharp‐edged high voltage tip, and ions produced are introduced into the mass spectrometer through a capillary. The EADESI technique can be applied to various samples including amino acids, peptides, proteins, drugs and human fluids such as urine and blood. EADESI is promising for routine analyses in different fields such as forensic, environmental and material sciences. EADESI interface can be fit to a conventional ion‐trap mass spectrometer. It can be used for various types of samples with a broad mass range. EADESI can also provide real‐time analysis which is very valuable for biomedical applications. Copyright © 2010 John Wiley & Sons, Ltd.