Efficient Protein Digestion Research Articles

Robust trypsin coating on electrospun polymer nanofibers in rigorous conditions and its uses for protein digestion

An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 and 50°C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 1.42 and 231 h at 40°C, respectively. The improved stability of EC-TR can be explained by covalent linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40°C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of 1 week. In the same condition, CA-TR showed poor performance from the beginning and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.

Trypsin Coatings on Electrospun and Alcohol-Dispersed Polymer Nanofibers for a Trypsin Digestion Column

The construction of a trypsin column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coatings, which were prepared by a two-step process of covalent attachment and enzyme cross-linking. In a comparative study with the trypsin coatings on as-spun and nondispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than 20 min. By applying the alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the trypsin coatings were improved, yielding more complete and reproducible digestions. Regardless of alcohol dispersion or not, trypsin coatings showed better digestion performance and improved performance stability under recycled uses than covalently attached trypsin, in-solution digestion, and commercial trypsin beads. The combination of highly stable trypsin coatings and alcohol dispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for online and automated protein digestion.

Prokaryotic Expression of Gly-Gln Dipeptide and Its Bioactive Analysis: A Novel Method for Short Peptide Production

Small bioactive peptides, with diverse biological functions, have received increasing attention as physiologically beneficial substances in animal production. The main obstacle to wide application of small bioactive peptides is a lack of cost-effective methods for mass production. In this study, we used glycyl-glutamine (Gly-Gln) as a test case to develop a novel mass-production method for small bioactive peptides. The oligonucleotide encoding Gly-Gln pro-peptide (Gpp) was designed and synthesized. Gpp includes 3 Gly-Gln dipeptides and 2 enzymatic sites for pepsin and trypase, allowing direct digestion and absorption of Gly-Gln in the gastrointestinal tract. The Gpp oligonucleotides were linked to generate an oligomeric oligonucleotide segment containing 12 tandem copies of Gpp. This 12Gpp segment was cloned and expressed in Escherichia coli vector pET32a. By optimizing culture conditions [0.1 mmol L −1 isopropyl-β-D-thiogalactopyranoside (IPTG), 50 μg mL −1 ampicillin (Amp), 30°C for 12 h], the thioredoxin fusion peptides reached 40% of total bacterial protein. After purification, the fusion protein was fed to Kunming mice to determine its effect on mouse immune function. The results showed that similar to Gly-Gln dipeptide, Gpp polymer protein could significantly suppress the proliferation of T and B lymphocytes in blood and spleen, and additionally could significantly improve interleukin-2 (IL-2) and interleukin-6 (IL-6) secretion of blood and spleen lymphocytes. These effects were not observed in mice fed a 2 amino acids mix (glycine and glutamine). These evidences indicated that an efficient digestion of Gpp polymer protein could be achieved when ingested into the animal gut. The expression system in this study provides a potential production method for not only Gly-Gln dipeptide but also other short bioactive peptides.

A Capillary Monolithic Trypsin Reactor for Efficient Protein Digestion inOnlineandOfflineCoupling to ESI and MALDI Mass Spectrometry

We describe the preparation of a capillary trypsin immobilized monolithic enzyme reactor (IMER) for a rapid and efficient digestion of proteins down to the femtomole level. Trypsin was immobilized on a poly(glycidyl methacrylate-co-acrylamide-co-ethylene glycol dimethycrylate) monolith using the glutaraldehyde technique. Digestion efficiencies of the IMER were evaluated using model proteins and protein mixtures as well as chemically cross-linked lysozyme regarding the addition of denaturants and increasing digestion temperature. The trypsin IMER described herein is applicable for the digestion of protein mixtures. Even at a 1000-fold molar excess of one protein, low-abundance proteins are readily identified, in combination with MS/MS analysis. An online setup of the IMER with reversed phase nano-HPLC separation and nano-ESI-MS/MS analysis was established. The great potential of the trypsin IMER for proteomics applications comprise short digestion times in the range of seconds to minutes, in addition to improved digestion efficiencies, compared to in-solution digestion.

Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes

In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.

Improvement of gel‐separated protein identification by DMF‐assisted digestion and peptide recovery after electroblotting

In-gel digestion of gel-separated proteins is a major route to assist in proteomics-based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in-digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel-separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (> or =99.8%). Before tryptic digestion, NH(4)HCO(3) buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane-bound substances, the proteins were virtually in-solution digested in DMF-containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane-enriched sample showed that the method was superior to the reported on-membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.

Comparisons of Mass Spectrometry Compatible Surfactants for Global Analysis of the Mammalian Brain Proteome

Methods for the global analysis of protein expression offer an approach to study the molecular basis of disease. Studies of protein expression in tissue, such as brain, are complicated by the need for efficient and unbiased digestion of proteins that permit identification of peptides by shotgun proteomic methods. In particular, identification and characterization of less abundant membrane proteins has been of great interest for studies of brain physiology, but often proteins of interest are of low abundance or exist in multiple isoforms. Parsing protein isoforms as a function of disease will be essential. In this study, we develop a digestion scheme using detergents compatible with mass spectrometry that improves membrane protein identification from brain tissue. We show the modified procedure yields close to 5,000 protein identifications from 1.8 mg of rat brain homogenate with an average of 25% protein sequence coverage. This procedure achieves a remarkable reduction in the amount of starting material required to observe a broad spectrum of membrane proteins. Among the proteins identified from a mammalian brain homogenate, 1897 (35%) proteins are annotated by Gene Ontology as membrane proteins, and 1225 (22.6%) proteins are predicted to contain at least one transmembrane domain. Membrane proteins identified included neurotransmitter receptors and ion channels implicated in important physiological functions and disease.

Digestion of Native Proteins for Proteomics Using a Thermocycler

Efficient protein digestion is a critical step for successful mass spectrometry analysis. Here we describe simultaneous tryptic digestion and gradual unfolding of native proteins by application of a temperature gradient using a single cycle of 5 min or less in a PCR thermocycler. Chemicals typically used for chromatographic techniques did not affect the digestion efficiency. Tryptic digestion was performed in a small volume (3 microL) with 1.5 microg of trypsin without denaturing agents. This rapid procedure yielded more peptides than conventional methods utilizing chemical denaturation for 18 proteins out of 20. Samples were directly spotted on the MALDI-TOF target plate, without additional purification, thus reducing losses on reversed-phase resins.

Development of an Automated Digestion and Droplet Deposition Microfluidic Chip for MALDI-TOF MS

An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 microm wide, 100 microm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 microL/min, producing a reaction time of approximately 24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, bovine serum albumin (BSA), myoglobin, and phosphorylase b, respectively.

Immobilization of trypsin in polyaniline-coated nano-Fe3O4/carbon nanotube composite for protein digestion

In this report, a four-component nanocomposite, trypsin-immobilized polyaniline-coated Fe3O4/carbon nanotube composite, was synthesized for highly efficient protein digestion. Fe3O4 was deposited by the chemical coprecipitation of Fe2+ and Fe3+ in an alkaline solution containing carbon nanotubes (CNTs) to prepare nano-Fe3O4/CNT composite. Subsequently, polyaniline (PA) was assembled on the Fe3O4/CNT composite by the in situ polymerization of aniline in the presence of trypsin to obtain trypsin-immobilized PA/Fe3O4/CNT nanocomposite. The novel 1D superparamagnetic biomaterial has been characterized by TEM, SEM, XRD, and magnetometric analysis. The feasibility and performance of the unique magnetic biomaterial have been demonstrated by the tryptic digestion of bovine serum albumin, myoglobin, and lysozyme within 5min. The digests were identified by MALDI-TOF MS with sequence coverages that were comparable to those obtained from the conventional in-solution tryptic digestion. The present biocomposite offers considerable promise for protein analysis due to its high magnetic responsivity and excellent dispersibility. It can be easily isolated from the digests with the aid of an external magnetic field. Because the enzyme-immobilized nanocomposite can be prepared by a simple two-step deposition approach at low cost, it may find a wide range of biological applications including proteome research.

High-Throughput Analysis of Rat Liver Plasma Membrane Proteome by a Nonelectrophoretic In-Gel Tryptic Digestion Coupled with Mass Spectrometry Identification

In-gel digestion is commonly used after proteins are resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the direct analysis of complex proteins. Here, we describe a strategy combining isolation of purified plasma membrane, efficient digestion of plasma membrane proteins in polyacrylamide gel, and high-sensitivity analysis by advanced mass spectrometry to create a new rapid and high-throughput method. The plasma membrane protein mixture is directly incorporated into a polyacrylamide gel matrix, After formation of the gel, proteins in the gel section are digested with trypsin, and the resulting peptides are subjected to reversed-phase, high-performance liquid chromatography followed by electrospray ion-trap tandem mass analysis. Using this optimized strategy, we have identified 883 rat liver membrane proteins, of which 490 had a gene ontology (GO) annotation indicating a cellular component, and 294 (60%) of the latter were known integral membrane proteins or membrane proteins. In total, 333 proteins are predicted by the TMHMM 2.0 algorithm to have transmembrane domains (TMDs) and 52% (175 of 333) proteins to contain 2-16 TMDs. The identified membrane proteins provide a broad representation of the rat plasma membrane proteome with little bias evident due to protein p I and molecular weight (MW). Also, membrane proteins with a high GRAVY score (grand average hydrophobicity score) were identified, and basic and acidic membrane proteins were evenly represented. This study not only offered an efficient and powerful method in shotgun proteomics for the identification of proteins of complex plasma membrane samples but also allowed in-depth study of liver membrane proteomes, such as of rat models of liver-related disease. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of rat liver plasma membrane in general.

A Nanoporous Reactor for Efficient Proteolysis

A nanoreactor based on mesoporous silicates is described for efficient tryptic digestion of proteins within the mesochannels. Cyano-functionalized mesoporous silicate (CNS), with an average pore diameter of 18 nm, is a good support for trypsin, with rapid in situ digestion of the model proteins, cytochrome c and myoglobin. The generated peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Proteolysis by trypsin-CNS is much more efficient than in-solution digestion, which can be attributed to nanoscopic confinement and concentration enrichment of the substrate within the mesopores. Proteins at concentrations of 2 ng muL(-1) were successfully identified after digestion for 20 min. A biological complex sample extracted from the cytoplasm of human liver tissue was digested by using the CNS-based reactor. Coupled with reverse-phase HPLC and MALDI-TOF MS/MS, 165 proteins were identified after standard protein data searching. This nanoreactor combines the advantages of short digestion time with retention of enzymatic activity, providing a promising way to advance the development of proteomics.

Using high‐concentration trypsin‐immobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature

We describe an innovative approach - using a high concentration of trypsin-modified magnetic nanoparticles (TMNPs) - for the rapid and efficient digestion of proteins at elevated temperature. The required digestion time could be reduced to less than 10 s. After digestion, the TMNPs were collected magnetically from the sample solution for reuse and the digested peptides were characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Protein digestion was optimized when using the TMNPs (5 microg/microL) at 57 degrees C; a significantly high peptide coverage was achieved for protein identification (e.g., 98% for lysozyme). Although a high concentration of TMNPs was used for digestion, the short digestion time led to much lower amounts of trypsin peptides being produced through self-digestion. As a result, interference in the mass spectrometric detection of the peptide ions was reduced significantly.

Rate of metabolic decarboxylation of leucine as assessed by a l[1‐13C1]leucine breath test combined with indirect calorimetry of broiler chickens fed isocaloric diets with different protein:fat ratio

Research has shown that broiler chickens reared on a low-protein diet have a more efficient protein digestion. However, information on the fate of absorbed amino acids in relation to the dietary crude protein level in poultry is sparse. Therefore, this study aimed at developing a methodology for a 1-(13)C(1)-leucine breath test combined with indirect calorimetry, and to apply this technique using broiler diets known to induce differences in protein retention. From 14 days of age onwards, broiler chickens were reared on one of two isocaloric diets with substitutions between fat and protein [low-protein (LP) vs. high-protein (HP) diet: 130.4 vs. 269 g protein/kg; and 101.8 vs. 27.9 g fat/kg]. Every 4 or 5 days, three chickens per diet were placed in the respiratory cells for 48 h. The broilers were intubated with 40 mg 1-(13)C(1)-leucine/kg body weight, followed by breath sampling for 4 h at 15-min intervals and mass spectrometric analysis of the (13)C:(12)C ratio in the samples. The CO(2) level in the respiratory cell air was monitored and excreta samples were collected. The methodology to study l[1-(13)C(1)]leucine decarboxyation in chickens using a breath test combined with indirect calorimetry was accomplished. Results of the nitrogen balance test indicated that the LP broilers had an improved dietary protein retention compared with the HP animals. Moreover, LP chickens decarboxylated a significantly lower percentage of l[1-(13)C(1)]leucine, demonstrating several 'protein- or amino acid-sparing' mechanisms in animals reared on a diet with lower protein level, both at the digestive and at the postabsorptive level.

Efficient protein digestion with peptide separation in a micro-device interfaced to electrospray mass spectrometry

A highly efficient protein digestion device has been fabricated using commercially available immobilized trypsin on agarose beads, packed into a silica capillary and connected either directly to an electrospray mass spectrometer via a 'microtight T' connector, from which aqueous acetic acid (0.2%) was pumped, or via a monolithic column connected to the mass spectrometer ion source. Six proteins with molecular mass ranging from 2848 to 77703 Da were digested completely using this system. In the second set of experiments a short monolithic separation column was placed after the immobilized trypsin capillary and partial separation of the generated peptides was obtained. The detection limits were increased from the micromol to pmol range by utilization of this separation column. Gradient elution, using a binary HPLC pump and a flow splitter, was used to optimize the peptide separation. This provided significantly enhanced resolution of the tryptic peptides but increased the analysis time to 30 minutes.

Zeolite nanoparticle modified microchip reactor for efficient protein digestion

An enzymatic microreactor has been fabricated based on the poly(methyl methacrylate) (PMMA) microchchip surface-modified with zeolite nanoparticles. By introducing the silanol functional groups, the surface of PMMA microchannel has been successfully modified with silicalite-1 nanoparticle for the first time due to its large external surface area and high dispersibility in solutions. Trypsin can be stably immobilized in the microchannel to form a bioreactor using silica sol-gel matrix. The immobilization of enzyme can be realized with a stable gel network through a silicon-oxygen-silicon bridge via tethering to those silanol groups, which has been investigated by scanning electron microscopy and microchip capillary electrophoresis with laser-induced fluorescence detection. The maximum proteolytic rate constant of the immobilized trypsin is measured to be about 6.6 mM s(-1). Using matrix assisted laser desorption and ionization time-of-flight mass spectrometry, the proposed microreactor provides an efficient digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL min(-1), which affords a very short reaction time of less than 5 s.

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 microg of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/microl or 37 fmol/microl. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein.

Digestive strategies in two sympatrically occurring lagomorphs

AbstractSeparation of low digestible fibres and fermentation of the digestible part of the food in the caecum is an adaptation of some small herbivores to cope with low‐quality forage. The caecum content is later re‐ingested as soft faeces so that the herbivore can benefit from this protein‐rich material. This is known as caecotrophy and is a common phenomenon in species of leporids, although differences exist between hares and rabbits. Hares have amorphous soft faeces and the amount of soft faeces produced is smaller compared to that of rabbits. Both factors suggest that hares have smaller benefits from re‐ingestion of the caecal contents compared with rabbits and, as a consequence, have a less efficient digestion (mainly of nitrogen) compared to rabbits. The assertion was tested whether digestive efficiency is different between the two herbivores and how this affects the choice of food plants in a natural situation. A feeding trial was conducted using hares and rabbits fed with diets with a range of fibre contents. Dry matter digestibility was not different, but nitrogen digestibility was lower in hares than in rabbits, indicating a less efficient digestion of protein. Both taxa showed a different response to increased fibre content in the diet. Rabbits maximized digestibility by increasing retention time of the food, hares maximized digestion rate by increasing the passage rate of the food through the digestive tract. The daily digestible nitrogen intake was higher in hares Lepus europaeus than that in rabbits Oryctolagus cuniculus, indicating that hares compensated for their lower nitrogen digestibility. Hares were predicted to select for higher quality plant species in a natural situation, but they had, on average, a lower nitrogen and higher total fibre content in their diet compared to sympatrically occurring rabbits. This indicated that hares did not compensate for their lower digestive efficiency by selecting higher quality food plants. The present experiment shows that hares and rabbits have different digestive strategies to cope with low quality forage. Rabbits had a higher N‐digestibility by increasing the retention time, whereas hares appeared to compensate for their lower N‐digestibility by increasing the processing rate, when food quality deteriorated.

Gene amplification and cold adaptation of pepsin in Antarctic fish. A possible strategy for food digestion at low temperature

Cold-adapted organisms have developed a number of adjustments at the molecular level to maintain metabolic functions at low temperatures. Among other features, they can produce enzymes characterized by a high turnover number or a high catalytic efficiency. The present work is aimed at investigating the process of food digestion at low temperature through the study of pepsins in Antarctic notothenioids. For such a purpose, we have cloned and sequenced three forms of pepsin A and a single form of gastricsin from the gastric mucosa of Trematomus bernacchii (rock cod). Phylogenetic analysis has suggested that the three pepsin A isotypes arose from two gene duplication events leading to the most ancestral pepsin A3 and to the most recent forms represented by pepsin A1 and pepsin A2. Molecular modeling has unraveled significant structural differences in these enzymes with respect to their mesophilic counterparts. Hydropathy and flexibility determined on the substrate-binding subsites of Antarctic and mesophilic pepsins have shown for pepsin A2 reduced hydropathy and increased flexibility at the level of the substrate cleft, features typical of cold-adapted enzymes. Northern blot analysis of RNA from rock cod gastric mucosa hybridized with molecular probes designed on specific regions of different pepsin forms has shown that rock cod pepsin genes are expressed at comparable levels. The present results suggest that the Antarctic rock cod adopted two different strategies to accomplish efficient protein digestion at low temperature. One mechanism is the gene duplication that increases enzyme production to compensate for the reduced kinetic efficiency, the other is the expression of a new enzyme provided with features typical of cold-adapted enzymes.

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping.

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.