Development of continuous microwave-assisted protein digestion with immobilized enzyme

In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC–MSⁿ. Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS–PAGE and LC–MSⁿ. Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC–MSⁿ profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research.     

Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

Benchmarking sample preparation/digestion protocols reveals tube‐gel being a fast and repeatable method for quantitative proteomics

Sample preparation, typically by in‐solution or in‐gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in‐gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS‐PAGE is a time‐consuming approach. Tube‐gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label‐free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label‐free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG‐prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841).     

Novel microwave-assisted digestion by trypsin-immobilized magnetic nanoparticles for proteomic analysis

In this study, a novel microwave-assisted protein digestion method was developed using trypsin-immobilized magnetic nanoparticles (TIMNs). The magnetic nanoparticles worked as not only substrate for enzyme immobilization, but also excellent microwave irradiation absorber and, thus, improved the efficiency of microwave-assisted digestion greatly. Three standard proteins, bovine serum albumin (BSA), myoglobin, and cytochrome c, were used to optimize the conditions of this novel digestion method. With the optimized conditions, peptide fragments produced in very short time (only 15 s) could be identified successfully by MALDI-TOF-MS. When it was compared to the conventional in-solution digestion (12 h), equivalent or better digestion efficiency was observed. Even when protein quantity was as low as micrograms, this novel digestion method still could digest proteins successfully, while the same samples by conventional in-solution digestion failed. Moreover, with an external magnetic field, the enzyme could be removed easily and reused. It was verified that, after 4 replicate runs, the TIMNs still kept high activity. To further confirm the efficiency of this rapid digestion method for proteome analysis, it was applied to the protein extract of rat liver. Without any preparation and prefractionation processing, the entire proteome digested by TIMNs in 15 s went through LC-ESI-MS/MS direct analysis. The whole shotgun proteomic experiment was finished in only 1 h with the identification of 313 proteins ( p < 0.01). This new application of TIMNs in microwave-assisted protein digestion really opens a route for large-scale proteomic analysis.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

Online microwave D-cleavage LC-ESI-MS/MS of intact proteins: site-specific cleavages at aspartic acid residues and disulfide bonds

An online nonenzymatic digestion method utilizing a microwave-heated flow cell and mild acid hydrolysis at aspartic acid (D) for rapid protein identification is described. This methodology, here termed microwave D-cleavage, was tested with proteins ranging in size from 5 kDa (insulin) to 67 kDa (bovine serum albumin) and a bacterial cell lysate ( Escherichia coli). A microwave flow cell consisting of a 5 microL total volume reaction loop connected to a sealed reaction vessel was introduced into a research grade microwave oven. With this dynamic arrangement, the injected sample was subjected to microwave radiation as it flowed through the reaction loop and was digested in less than 5 min. Different digestion times can be achieved by varying the sample flow rate and/or length of the loop inside the microwave flow cell. The microwave flow cell can be operated individually with the output being collected for matrix assisted laser ionization/desorption (MALDI) mass spectrometry (MS) or connected online for liquid chromatography (LC) electrospray ionization (ESI)-MS. In the latter configuration, the microwave flow cell eluates containing digestion products were transferred online to a reversed phase liquid chromatography column for direct ESI-MS and ESI-MS/MS analyses (specifically, Collision Induced Dissociation, CID). Concurrently with the microwave D-cleavage step, disulfide bond reduction/cleavage was achieved by the coinjection of dithiothreitol (DTT) with the sample prior to online microwave heating and online LC-MS analysis and so eliminating the need for alkylation of the reduced protein. All protein standards, protein mixtures, and proteins in a bacterial cell lysate analyzed by this new online methodology were successfully identified via a SEQUEST database search of fragment ion mass spectra. Overall, online protein digestion and identification was achieved in less than 40 min total analysis time, including the chromatographic step.     

Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing

We have investigated the use of a variety of different techniques to identify as many proteins as possible in a yeast lysate, with the aim of investigating the overlap and complementarity of data from different approaches. A standard lysate was prepared from log phase yeast (Saccharomyces cerevisiae). This was then subjected to analysis via five different approaches aimed at identifying as many proteins as possible using an ion trap mass spectrometer. The total number of non-redundant protein identifications from each experiment was: 524 proteins by 2-D (SCX/C18) nanoflow liquid chromatography-liquid chromatography tandem mass spectrometry (nanoLC-LC MS/MS (MudPIT)); 381 proteins by nanoLC-MS/MS with gas phase fractionation by mass range selection; 390 proteins by nanoLC-MS/MS with gas phase fractionation by ion abundance selection; 898 proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins, in-gel digestion, and nanoLC-MS/MS of gel slices; and 422 proteins by isoelectric focusing of proteins, in-gel digestion and nanoLC-MS/MS of gel slices. The total number of non-redundant protein identifications in the five experiments was 1204. Combining only the two best experiments, the SDS-PAGE gel slices and the Mudpit, produces 1024 proteins identified, more than 85% of the total. Clearly, combining a Mudpit analysis with an SDS-PAGE gel slice experiment gives the greatest amount of protein identification information from a limited amount of sample.     

Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler

A completely automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system for characterization of therapeutic proteins in which a common high-performance liquid chromatography (HPLC) autosampler is used for automated sample preparation, including protein denaturation, reduction, alkylation, and enzymatic digestion, is described. The digested protein samples are then automatically subjected to LC/MS analysis using the same HPLC system. The system was used for peptide mapping of monoclonal antibodies (mAbs), known as a challenging group of therapeutic proteins for achieving complete coverage and quantitative representation of all peptides. Detailed sample preparation protocols, using an Agilent HPLC system, are described for Lys-C digestion of mAbs with intact disulfide bonds and tryptic digestion of mAbs after reduction and alkylation. The automated procedure of Lys-C digestion of nonreduced antibody, followed by postdigestion disulfide reduction, produces both the nonreduced and reduced digests that facilitate disulfide linkage analysis. The automated peptide mapping LC/MS system has great utility in preparing and analyzing multiple samples for protein characterization, identification, and quantification of posttranslational modifications during process and formulation development as well as for protein identity and quality control.     

A new application of microwave technology to proteomics

Two-dimensional electrophoresis (2-DE) combined with mass spectrometry has significantly improved the possibilities of large-scale identification of proteins. However, 2-DE is limited by its inability to speed up the in-gel digestion process. We have developed a new approach to speed up the protein identification process utilizing microwave technology. Proteins excised from gels are subjected to in-gel digestion with endoprotease trypsin by microwave irradiation, which rapidly produces peptide fragments. The peptide fragments were further analyzed by matrix-assisted laser desorption/ionization technique for protein identification. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several proteins, including lysozyme, albumin, conalbumin, and ribonuclease A. The method reduced the required time for in-gel digestion of proteins from 16 hours to as little as five minutes. This new application of microwave technology to protein identification will be an important advancement in biotechnology and proteome research.     

An optimized protein in-gel digest method for reliable proteome characterization by MALDI-TOF-MS analysis

Human lung epithelial cells (A549) were used as a model to develop a reliable proteome characterization method by peptide mass fingerprinting (PMF). Lung cell lysate proteins and protein standards were separated by 2D-gel electrophoresis, stained with Coomassie blue, gel plugs were subjected to commonly adapted as well as optimized in-gel digestion/sample preparation methods. Samples were analyzed by MALDI-TOF-MS. Optimization parameters included, use of NH(4)OAc in destaining and in-gel digestion buffers, detergent/salt removal prior to in-gel digestion, use of solvents of varying polarities (0%, 30%, 60% ACN containing 0.1% TFA) to improve peptide recoveries, matrix composition (alpha-cyano-4-hydroxycinamic acid-organic solvent combinations) and on-target salt removal. This led to enhanced mass spectral information and a sensitivity gain in the order of 6-10 fold compared to that of common procedures, yielding reliable, unambiguous protein identification with femtomol protein sensitivity by Autoflex MALDI-TOF-MS. Triplicate analyses by two analysts revealed consistent, wide range m/z values including in < 1200Da region by relieving matrix-exerted signal suppression, requiring one trial to obtain a unique protein identification with superior PMF results for the optimized method. Analyses of ten A549 proteins in replicates using the optimized method yielded fast, reliable characterization, suggesting the potential application of this method in high-throughput protein identification by PMF.     

Increased proteome coverage by combining PAGE and peptide isoelectric focusing: Comparative study of gel‐based separation approaches

The in‐depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC‐MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in‐gel IEF, prior to RP‐HPLC‐MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.     

Direct atmospheric pressure coupling of polyacrylamide gel electrophoresis to mass spectrometry for rapid protein sequence analysis

Using laser desorption-atmospheric pressure chemical ionization we describe a novel approach for coupling mass spectrometry to polyacrylamide gel electrophoresis. In contrast to other approaches, the method allows for the direct sampling of a polyacrylamide gel-embedded protein without the addition of any exogenous matrixes and is performed at atmospheric pressure. After electrophoresis and enzymatic digestion, the gel is analyzed at AP by photons that desorb neutral peptide molecules, followed by corona discharge ionization in the gas-phase, and subsequent mass analysis. Our experimental results demonstrate the method to (1) rapidly identify electrophoresed proteins via "peptide fingerprinting" using protein databases, (2) detect single-amino acid polymorphisms, and (3) has potential for sub-picomole sensitivity while still maintaining in situ gel desorption-ionization at ambient conditions.     

1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome

Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.     

Liquid-chromatographic and mass-spectrometric identification of lens proteins using microwave-assisted digestion with trypsin-immobilized magnetic nanoparticles

We used a newly developed method combining trypsin-immobilized magnetic nanoparticles (TIMNs) and microwave-assisted protein digestion to study the proteins of human lens tissue. The digested proteins were identified by liquid chromatography and mass spectrometry. The lens proteins were digested under optimized conditions (digestion time 1 min, microwave power 400 W, trypsin-to-protein ratio 1:5) determined using bovine serum albumin as the standard protein, before liquid-chromatographic and mass-spectrometric analysis. Twenty-six proteins were identified with the new digestion method compared with 11 proteins identified with traditional in-solution digestion (12 h). γ-Crystallin, β-crystallin, and superoxide dismutase 1 proteins, identified with the microwave-assisted method but not the traditional method, are related to cataract development according to some studies. The TIMNs were easily separated from the digestion products. This new digestion method could prove extremely useful for large-scale proteomic analyses.     

Mass spectrometry for the study of protein-protein interactions

The identification of subpicomolar amounts of protein by mass spectrometry (MS) coupled with two-dimensional methods to separate complex protein mixtures is fueling the field of proteomics, and making feasible the notion of cataloging and comparing all of the expressed proteins in a biological sample. Functional proteomics is a complementary effort aimed at the characterization of functional features of proteins, such as their interactions with other proteins. Proteins comprise modular domains, many of which are noncatalytic modules that direct protein-protein interactions. Capturing proteins of interest and their interacting proteins by using high-affinity antibodies presents a simple method to prepare relatively simple protein mixtures easily resolved in one-dimensional formats. Individual or mixtures of proteins identified as stained bands in polyacrylamide gels are subjected to in situ digestion with the protease trypsin, and the extracted peptide fragments are analyzed by MS. The quality, quantity, and complexity of the tryptic digest, the species origin of the proteins, and the quality of the corresponding databases of genomic and protein information greatly influence the subsequent MS analysis in terms of degree of difficulty and methodological approach required to make an unambiguous protein identification. In this article we report the isolation of associated proteins from a complex cell-derived lysate by using an epitope-directed antibody. The protein pICLn engineered to carry an epitope tag was purified from cultured human embryonic kidney cells, and found to associate with a variety of proteins including the spliceosomal proteins smE and smG. By application of this general approach, the systematic identification of protein complexes and assignment of protein function are possible.     

Comparative study of [Three] LC-MALDI workflows for the analysis of complex proteomic samples

Large-scale proteomic analyses frequently rely on high-resolution peptide separation of digested protein mixtures in multiple dimensions to achieve accuracy in sample detection and sensitivity in dynamic range of coverage. This study was undertaken to demonstrate the feasibility of MALDI MS/MS with off-line coupling to HPLC for the analysis of whole cell lysates of wild-type yeast by three different workflows: SCX-RPHPLC-MS/MS, high-pH SAX-RPHPLC-MS/MS and RP (protein)-SCX-RPHPLC-MS/MS. The purpose of these experiments was to demonstrate the effect of a workflow on the end results in terms of the number of proteins detected, the average peptide coverage of proteins, and the number of redundant peptide sequencing attempts. Using 60 microg of yeast lysate, minor differences were seen in the number of proteins detected by each method (800-1200). The most significant differences were observed in redundancy of MS/MS acquisitions.     

Limited acid hydrolysis as a means of fragmenting proteins isolated upon ProteinChip array surfaces

ProteinChip array technology enables protein purification, protein profiling, and biomarker discovery on a convenient biochip platform. Traditional proteomic approaches towards protein identification rely upon the generation of peptides through the use of specific proteases. However, for a variety of reasons, the digestion of proteins bound to planar arrays by specific proteases, such as trypsin, has proven to be difficult, at times providing little or no protein digestion at all. Additionally, should more than one protein be present on the array surface, the digestion product consists of peptides from different proteins, adding another dimension of complexity to database mining approaches. These factors have driven our group to explore alternative means of on-chip protein digestion. In this article, we describe an approach to generate peptide maps by limited acid hydrolysis. Depending upon the adsorbed protein, this method requires between 500 femtomole to 5 picomole of protein for on-chip hydrolysis. Besides generating several internal peptide fragments, limited acid hydrolysis also has the advantage of generating peptide ladders from the N- or C-terminus of the protein. From these ladders, partial primary sequence of the protein can be directly derived when analyzed by a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry can be performed on several internal peptide fragments, thus facilitating the identification of several proteins within a mixture. Based upon the preliminary results of this work, we continue to explore the possibility of using limited acid hydrolysis to identify unknown proteins captured on ProteinChip array surfaces.     

Proteome profile of human breast cancer tissue generated by LC-ESI-MS/MS combined with sequential protein precipitation and solubilization

Comprehensive proteome profiling of breast cancer tissue samples is challenging, as the tissue samples contain many proteins with varying concentrations and modifications. We report an effective sample preparation strategy combined with liquid chromatography (LC) electrospray ionization (ESI) quadrupole time-of-flight (QTOF) MS/MS for proteome analysis of human breast cancer tissue. The complexity of the breast cancer tissue proteome was reduced by using protein precipitation from a tissue extract, followed by sequential protein solubilization in solvents of different solubilizing strength. The individual fractions of protein mixtures or subproteomes were subjected to trypsin digestion and the resultant peptides were separated by strong cation exchange (SCX) chromatography, followed by reversed-phase capillary LC combined with high resolution and high accuracy ESI-QTOF MS/MS. This approach identified 14407 unique peptides from 3749 different proteins based on peptide matches with scores above the threshold scores at the 95% confidence level in MASCOT database search of the acquired MS/MS spectra. The false positive rate of peptide matches was determined to be 0.95% by using the target-decoy sequence search strategy. On the basis of gene ontology categorization, the identified proteins represented a wide variety of biological functions, cellular processes, and cellular locations.     

Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry

The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed-phase (RP) microcolumns prior to mass spectrometric analysis have resulted in increased signal-to-noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation on microcolumns, or are suppressed in the ionization process. We report here the use of graphite powder packed in constricted GELoader tips as an alternative to RP microcolumns for desalting and concentration of peptide mixtures prior to MS. Such columns are able to retain small and/or hydrophilic peptides that can be lost when using RP microcolumns. In addition, we show that samples contaminated with small biological polymers can readily be analyzed using graphite powder rather than RP microcolumns, since the polymer molecules bind strongly to graphite and are not eluted with the peptides.     

Can electromagnetic fields influence the structure and enzymatic digest of proteins? A critical evaluation of microwave-assisted proteomics protocols

This study reevaluates the putative advantages of microwave-assisted tryptic digests compared to conventionally heated protocols performed at the same temperature. An initial investigation of enzyme stability in a temperature range of 37-80°C demonstrated that trypsin activity declines sharply at temperatures above 60°C, regardless if microwave dielectric heating or conventional heating is employed. Tryptic digests of three proteins of different size (bovine serum albumin, cytochrome c and β-casein) were thus performed at 37°C and 50°C using both microwave and conventional heating applying accurate internal fiber-optic probe reaction temperature measurements. The impact of the heating method on protein degradation and peptide fragment generation was analyzed by SDS-PAGE and MALDI-TOF-MS. Time-dependent tryptic digestion of the three proteins and subsequent analysis of the corresponding cleavage products by MALDI-TOF provided virtually identical results for both microwave and conventional heating. In addition, the impact of electromagnetic field strength on the tertiary structure of trypsin and BSA was evaluated by molecular mechanics calculations. These simulations revealed that the applied field in a typical laboratory microwave reactor is 3-4 orders of magnitude too low to induce conformational changes in proteins or enzymes.