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.     

Silaffin Peptides as a Novel Signal Enhancer for Gravimetric Biosensors

Application of biomimetic silica formation to gravimetric biosensors has been conducted for the first time. As a model system, silaffin peptides fused with green fluorescent protein (GFP) were immobilized on a gold quartz crystal resonator for quartz crystal microbalances using a self-assembled monolayer. When a solution of silicic acid was supplied, silica particles were successfully deposited on the Au surface, resulting in a significant change in resonance frequency (i.e., signal enhancement) with the silaffin–GFP. However, frequency was not altered when bare GFP was used as a control. The novel peptide enhancer is advantageous because it can be readily and quantitatively conjugated with sensing proteins using recombinant DNA technology. As a proof of concept, this study shows that the silaffin domains can be employed as a novel and efficient biomolecular signal enhancer for gravimetric biosensors.     

Affinity Chromatography of Human Blood Coagulation Factor VIII on Monoliths with Peptides from a Combinatorial Library

FVIII is a very complex molecule of great therapeutic significance. It is purified by a sequence of chromatographic steps including immunoaffinity chromatography. A peptide affinity chromatography method has been developed using peptides derived from a combinatorial library. Spot technology using cellulose sheets has been applied for this purpose. The dual positional scanning strategy was used for identification of the amino acids in random positions. Approximately 5000 possible candidates found in the first screening round were reduced to a panel of 36. Six candidates have been selected empirically. Five peptides seem to be directed against the light chain of FVIII, one peptide seems to be directed against the heavy chain. The peptides have been immobilized on conventional beaded material and CIM polymethacrylate monoliths. Much better performance with respect to capacity and selectivity has been observed with the monolithic material. Exposure of the ligand and its ensuing accessibility are responsible for these properties.     

Average peptide score: a useful parameter for identification of proteins derived from database searches of liquid chromatography/tandem mass spectrometry data

The quantity and variable quality of data that can be generated from liquid chromatography (LC)/mass spectrometry (MS)-based proteomics analyses creates many challenges in interpreting the spectra in terms of the actual proteins in a complex sample. In spite of improvements in algorithms that match putative peptide sequences to MS/MS spectra, the assembly of these lists of possible or probable peptides into a 'correct' set of proteins is still problematic. We have observed a trend in a simple relationship, derived from standard database search outputs, which can be useful in assessing the quality of a MS/MS-based protein identification. Specifically, the ratio of the protein score and number of non-redundant peptides, or average peptide score (APS), can facilitate initial filtering of database search results in addition to providing a useful measure of confidence for the proteins identified. This parameter has been applied to results from the analysis of multi-protein complexes derived from pull-down experiments analyzed using a two-dimensional LC/MS/MS workflow. In particular, the complex list of protein identifications derived from a drug affinity pull-down with immobilized ampicillin and an E. coli lysate was greatly simplified by applying the APS as a filter, allowing for facile identification of the penicillin-binding proteins known to interact with ampicillin. Furthermore, an APS threshold can be used for any data sets derived from electrospray ionization (ESI)- or matrix-assisted laser desorption/ionization (MALDI)-MS/MS experiments and is also not specific to any database search program.     

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.     

Plant pathogen recognition as a natural, original and simple model for chemogenomics: a brief overview of cell-based assays to screen for peptides acting as plant defense activators

As plants lack a circulatory system and adaptive immune system, they have evolved their own defense systems distinct from animals, in which each plant cell is capable of defending itself from pathogens. Plants induce a number of defense responses, which are triggered by a variety of molecules derived from pathogenic microorganisms, referred to as microbe-associated molecular patterns (MAMPs), including peptides, proteins, lipopolysaccharide, beta-glucan, chitin, and ergosterol. The interaction between plants and chemicals in the context of plant defense represents a "natural" and simple model for chemogenomics, at the intersection between chemical and biological diversities. For protection of crop plants from diseases, it has been shown to be effective to stimulate the plant immunity by chemical compounds, the so-called "plant defense activators". Combinatorial chemistry techniques can be applied to the search for novel plant defense activators, but it is essential to establish an efficient and reliable screening system suitable for library screening. For studies of the plant immune system, it is difficult to use isolated proteins as biological targets because the receptors for MAMP recognition are largely unknown and even the receptors identified so far are transmembrane proteins. Therefore, screening for novel peptides acting on MAMP receptors from combinatorial libraries must rely on a solution-phase assay using cells as the biological targets. In this review, we introduce the cell-based lawn format assay for identification of peptides acting as plant defense activators from combinatorial peptide libraries. The requirements and limitations in constructing the screening system using combinatorial libraries in the studies of plant sciences are also discussed.     

Optimization of immunoaffinity enrichment and detection: toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry

Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.     

Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis

The design of an extended-run 96-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and the development of protein detection technology based upon fluorescein derivatives that bind to peptide epitope tags, allows the creation of a truly high-throughput analysis of protein expression, where less than 20 min are needed to separate proteins and analyze results. We demonstrate the overall capabilities of such a method combination in a complex cell lysate background, while comparing the specific results obtained using a biarsenical fluorescein-derivative and tetracysteine epitope-tagged proteins with total protein staining using a fluorescent gel stain and with Western blotting where an anti-oligohistidine (His) tag antibody has been employed. When applied on purified target proteins without extraneous protein background, the demonstrated sensitivity of the assay on the extended-run 96-array precast SDS-PAGE system allows detection of quantities of tagged protein as low as 1 pmol per band.     

A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity.

A novel multiphasic buffer system for high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively.     

Oligomerization of uniquely folded mini-protein motifs: development of a homotrimeric betabetaalpha peptide.

The discovery of a discretely folded homotrimeric betabetaalpha motif (BBAT1) was recently reported (J. Am. Chem. Soc. 2001, 123, 1002-1003). Herein the design, synthesis, and analysis of a small library of peptides which led to the isolation of BBAT1 is described. betabetaalpha peptides based on the monomeric sequence of BBA5 (Folding Des. 1998, 120, 95-103) were synthesized to include the anthranilic acid/nitrotyrosine fluorescence quenching pair to rapidly detect interpeptide association. In the first generation of peptides synthesized, truncations in the loop region connecting the beta-hairpin to the alpha-helix revealed that a two-residue deletion in the loop promoted an interpeptide association as detected by fluorescence quenching. An additional library of 22 loop-truncated betabetaalpha peptides was subsequently synthesized to include a variety of sequence mutations in an effort to enhance the observed peptide-peptide binding. From the fluorescence quenching screen, peptide B2 was found to possess the strongest fluorescence-quenching response, indicative of a strong peptide-peptide association. Due the poor solubility of peptide B2, the S-methylated cysteine at position 9 in the loop was substituted with a glycine to generate peptide BBAT1 which possessed greatly improved water solubility and formed discrete trimers. The successful design of this oligomeric betabetaalpha structure will likely aid the design of more complex alpha-beta superstructures and further our understanding of the factors controlling protein-protein interactions at alpha-beta protein interfaces.     

Synthesis of a novel macrocyclic library: discovery of an IGF-1R inhibitor

We present a new approach for the conversion of active sequences of proteins and peptides into small molecules. A library of macrocyclic disulfide molecules was made, in which the active pharmacophores of the parent peptide are preserved while the size of the macromolecular scaffold on which the pharmacophores are arranged is varied. This enables a systematic search for macromolecules in which the pharmacophores are in an appropriate conformation for biological activity. We developed two procedures for the synthesis of such libraries from building blocks that include commercial amino acids and functionalized aldehydes. Chemical synthesis using the "tea-bag" method gave a library with higher diversity, but low yields, compared to the manual synthesis of the library, in which the compounds were synthesized in individual vessels and the yield and purity improved dramatically. As a proof of concept, we synthesized a 34-member library derived from the sequence of the activation loop of insulin-like growth factor-1 receptor. Selected compounds were screened, and one was found to be biologically active in the low micromolar range. The concept presented here may prove particularly useful in cases where the pharmacophores are known but need to be systematically screened for a spatial arrangement that will enable biological activity.     

India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with Western blots for peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present an approach that allows matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) peptide mapping of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (NC). After blocking the nitrocellulose membrane with polyvinylpyrrolidone-40 the immobilized proteins are visualized using India Ink staining which allows the detection of low nanogram amounts of protein. The utilization of a low concentration of Tween 20 (0.05%) in the India Ink staining solution does not negatively impair the quality of the mass spectra. Due to the virtual nondestructive nature of the stain proteolytic peptides could be recovered from the NC membrane. Taking into account minor precautions during the sample manipulation and concentration and by loading the sample onto a pre-crystallized matrix layer, high quality mass spectral data were obtained on <100 femtomoles of protein loaded onto the gel. Finally, the use of India Ink in conjunction with Western blot analysis is also demonstrated. A rat plasma protein, characterized by Western blot as a covalently modified protein-drug compound, was subjected to peptide mapping and post source decay (PSD) sequencing of peptides. The zomepirac-modified protein was identified as the alpha-subunit of fibrinogen.     

Analysis of the low molecular weight serum peptidome using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer

Advances in proteomics are continuing to expand the ability to analyze the serum proteome. In recent years, it has been realized that in addition to the circulating proteins, human serum also contains a large number of peptides. Many of these peptides are believed to be fragments of larger proteins that have been at least partially degraded by various enzymes such as metalloproteases. Identifying these peptides from a small amount of serum/plasma is difficult due to the complexity of the sample, the low levels of these peptides, and the difficulties in getting a protein identification from a single peptide. In this study, we modified previously published protocols for using centrifugal ultrafiltration, and unlike past studies did not digest the filtrate with trypsin with the intent of identifying endogenous peptides with this method. The filtrate fraction was concentrated and analyzed by a reversed phase-high performance liquid chromatography system connected to a nanospray ionization hybrid ion trap-Fourier transform mass spectrometer (LTQ-FTMS). The mass accuracy of this instrument allows confidence for identifying the protein precursors by a single peptide. The utility of this approach was demonstrated by the identification of over 300 unique peptides with 2 ppm or better mass accuracy per serum sample. With confident identifications, the origin and function of native serum peptides can be more seriously explored. Interestingly, over 34 peptide ladders were observed from over 17 serum proteins. This indicates that a cascade of proteolytic processes affects the serum peptidome. To examine whether this result was an artifact of serum, matched plasma and serum samples were analyzed with similar peptide ladders found in each.     

Mass spectrometric detection of affinity purified crosslinked presented

Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest of the products shows considerable promise as a tool for interrogating structure and geometry of proteins and protein complexes. An impediment to the use of this tool has been the difficulty of distinguishing crosslinked peptide pairs from non-crosslinked peptides, and from the products of side reactions. We describe the use of a commercially available biotinylated crosslinking reagent, sulfo-SBED, that allows affinity-based enrichment of crosslinked species. An intramolecular crosslink is prepared using the peptide neurotensin as a model system. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra show the predicted crosslinking product, as well as several side products. Finally, we describe the optimized enrichment of biotinylated species, and reduction of non-specific binding, for a batch-mode affinity separation based on immobilized monomeric avidin.     

Rational design of affinity peptide ligand by flexible docking simulation

Rational design of affinity peptide ligands of proteins by flexible docking simulation is performed using the SYBYL program package. This approach involves the use of experimental data to verify a scoring function that can be used to assess the affinity of a peptide for its target protein. The enzyme-linked immunosorbent assay (ELISA) data of several peptides displayed on phage surfaces for insulin and lysozyme, respectively, reported in literature are used for the purpose. It is found that the absolute values of the Dscore calculated from the docking correspond well to the ELISA data that relate to the affinity between the peptides and the target molecule. So, the Dscore function is used to assess the affinity of docked peptides in a pentapeptide library designed on the basis of protein (alpha-amylase) structure. As a result, a pentapeptide with a high Dscore value is selected and a hexapeptide (FHENWS) is built by linking serine to its C-terminal to lengthen the peptide. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions (including hydrogen bonds) and Van der Waals forces contribute to the affinity of the hexapeptide for alpha-amylase. Chromatographic experiments with the immobilized peptide have given further evidence for this observation. Adsorption isotherm described by the Langmuir equation indicates that the apparent binding constant of alpha-amylase to the immobilized hexapeptide was 2.5x10(5)L/mol. Finally, high affinity and specificity of the affinity adsorbent is exemplified by the purification of alpha-amylase from crude fermentation broth of Bacillus subtilis.     

Digestion of Enolase and Carbonic Anhydrase as Model Proteins for Therapeutic Proteins in Blood Plasma with Immobilized Thermolysin and Quantification of Some of the Peptides by LC/LC–MS/MS

There is a need for fast method development in the early drug discovery phase of therapeutic proteins. Thermolysin has not been used for quantification of proteins in blood plasma earlier. It is a thermostable protease which permits the use of high temperatures for fast hydrolysis of proteins. Model proteins were digested with immobilized thermolysin on agarose gel. Protein-specific peptides were selected for quantitation and quantified based on stable isotope dilution. Protein digests of blood plasma were cleaned and separated using an automated LC/LC–MS/MS system. Essential digestion parameters that influence thermolysin hydrolytic activity were optimized for high peptide yield. The validated methods were selective, linear, precise and accurate with a limit of detection of 2 nM for both proteins. The proposed strategy for method development could be valuable for quantification of proteins in blood plasma samples. The study underscores and discusses important features of the enzymatic digestion and chromatographic separation.     

Identification of Amino Acid Residues Responsible for the Binding to Anti-FLAG™ M2 Antibody Using a Phage Display Combinatorial Peptide Library

FLAG, a short hydrophilic peptide consisting of eight amino acids (DYKDDDDK), has been widely used as a fusion tag for the purification and detection of a wide variety of recombinant proteins. One of the monoclonal antibodies against this peptide, anti-FLAG M2, recognises a FLAG peptide sequence at the N terminus, Met-N terminus, C terminus, or internal site of a fusion protein and has been extremely useful for the detection, identification, and purification of recombinant proteins. Nevertheless, detailed binding specificity of anti-FLAG M2 has yet to be determined. In the current study, a phage display combinatorial peptide library was used to determine that the motif DYKxxD encompasses the critical amino acid residues responsible for the binding of FLAG peptide to this antibody. This study demonstrates the utility of phage display technology and helps to elucidate the mode of action of this detection system.     

Histidine‐Containing Peptide Catalysts Developed by a Facile Library Screening Method

Although peptide catalysts have a high potential for the use as organocatalysts, the optimization of peptide sequences is laborious and time‐consuming. To address this issue, a facile screening method for finding efficient aminocatalysts from a peptide library has been developed. In the screening for the Michael addition of a malonate to an enal, a dye‐labeled product is immobilized on resin‐bound peptides through reductive amination to visualize active catalysts. This procedure allows for the monitoring of the reactivity of entire peptides without modifying the resin beads beforehand. Peptides containing histidine at an appropriate position were identified by this method. A novel function of the histidyl residue, which enhances the binding of a substrate to the catalyst by capturing an iminium intermediate, was indicated.     

A peptide microarray for the detection of protein kinase activity in cell lysate.

DNA microarray enables the analysis of DNA or mRNA expression levels, but it has not been possible to completely understand life using obtained information. Consequently, protein or peptide arrays have attracted much interest. Since the development of a practical protein microarray is still far away in light of handling difficulties, the peptide microarray is a promising tool for analyzing protein functions. We have developed a peptide microarray to detect protein kinase activity in cell lysate. All substrate peptides for kinases were immobilized chemoselectively on amino-coated glass slides. After phosphorylation of the immobilized peptides, phosphorylation was detected by fluorescence imaging. We detected the protein kinase activities, including that in cell lysate, in response to drug stimulation. Therefore, this peptide microarray would be useful for a high-throughput kinase assay of intracellular signals and would be applicable to drug screening.     

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.