Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor

Nanopore technology has become a highly sensitive and powerful tool for single molecule sensing of chemicals and biopolymers. Protein pores have the advantages of size amenability, channel homogeneity, and fabrication reproducibility. But most well‐studied protein pores for sensing are too small for passage of peptide analytes that are typically a few nanometers in dimension. The funnel‐shaped channel of bacteriophage phi29 DNA packaging motor has previously been inserted into a lipid membrane to serve as a larger pore with a narrowest N‐terminal constriction of 3.6 nm and a wider C‐terminal end of 6 nm. Here, the utility of phi29 motor channel for fingerprinting of various peptides using single molecule electrophysiological assays is reported. The translocation of peptides is proved unequivocally by single molecule fluorescence imaging. Current blockage percentage and distinctive current signatures are used to distinguish peptides with high confidence. Each peptide generated one or two distinct current blockage peaks, serving as typical fingerprint for each peptide. The oligomeric states of peptides can also be studied in real time at single molecule level. The results demonstrate the potential for further development of phi29 motor channel for detection of disease‐associated peptide biomarkers.     

Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching

A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.     

Recognition of dengue virus protein using epitope-mediated molecularly imprinted film

Molecularly imprinted film was fabricated in the presence of a pentadecapeptide onto a quartz crystal microbalance (QCM) chip. This 15-mer peptide has been known as the linear epitope of the dengue virus NS1 protein. Imprinting resulted in an increased polymer affinity toward the corresponding templates but also to the virus protein. Direct detection of the dengue virus protein was achieved quantitatively. The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies. The binding effect was further enhanced and confirmed using a monoclonal antibody to form a sandwich with the MIP-NS1 protein complex on the chip. No pretreatment was required.     

Rational selection of the optimum MALDI matrix for top-down proteomics by in-source decay

In-source decay (ISD) in MALDI leads to c- and z-fragment ion series enhanced by hydrogen radical donors and is a useful method for sequencing purified peptides and proteins. Until now, most efforts to improve methods using ISD concerned instrumental optimization. The most widely used ISD matrix is 2,5-dihydroxybenzoic acid (DHB). We present here a rational way to select MALDI matrixes likely to enhance ISD for top-down proteomic approaches. Starting from Takayama's model (Takayama, M. J. Am. Soc. Mass Spectrom. 2001, 12, 1044-9), according to which formation of ISD fragments (c and z) would be due to a transfer of hydrogen radical from the matrix to the analyte, we evaluated the hydrogen-donating capacities of matrixes, and thus their ISD abilities, with spirooxazines (hydrogen scavengers). The determined hydrogen-donating abilities of the matrixes are ranked as follows: picolinic acid (PA) > 1,5-diaminonaphtalene (1,5-DAN) > DHB > sinapinic acid > alpha-cyano-4-hydroxycinnamic acid. The ISD enhancement obtained by using 1,5-DAN compared to DHB was confirmed with peptides and proteins. On that basis, a matrix-enhanced ISD approach was successfully applied to sequence peptides and proteins up to approximately 8 kDa. Although PA alone is not suitable for peptide and protein ionization, ISD signals could be further enhanced when PA was used as an additive to 1,5-DAN. The optimized matrix preparation was successfully applied to identify larger proteins by large ISD tag researches in protein databases (BLASTp). Coupled with an adequate separation method, ISD is a promising tool to include in a top-down proteomic strategy.     

On the utility of isotopic fine structure mass spectrometry in protein identification

Modern mass spectrometry (MS)-based protein identification and characterization relies upon accurate mass measurements of the (13)C isotopic distributions of the enzymatically produced peptides. Interestingly, obtaining peptide elemental composition information from its isotopic fine structure mass spectrum to increase the confidence in peptide and protein identification has not yet been developed into a bottom-up proteomics-grade analytical approach. Here, we discuss the possible utility and limitations of the isotopic fine structure MS for peptide and protein identification. First, we in silico identify the peptides from the E. coli tryptic digest and show the increased confidence in peptide identification by consideration of the isotopic fine structures of these peptides as a function of mass and abundance accuracies. In the following, we demonstrate that the state-of-the-art high magnetic field Fourier transform ion cyclotron resonance (FT-ICR) MS allows a routine acquisition of the isotopic fine structure information of a number of isobaric peptide pairs, including a pair of peptides originating from E. coli. Finally, we address the practical limitation of the isotopic fine structure MS implementation in the time-constraint experiments by applying an advanced signal processing technique, filter diagonalization method, to the experimental transients to overcome the resolution barrier set by the typically applied Fourier transformation. We thus demonstrate that the isotopic fine structures of peptides may indeed improve the peptide and possibly protein identification, can be produced in a routine experiment by the state-of-the-art high resolution mass spectrometers, and can be potentially obtained on a chromatographic time-scale of a typical bottom-up proteomics experiment. The latter one requires at least an order of magnitude increase in sensitivity of ion detection, which presumably can be realized using high-field Orbitrap FTMS and/or future generation of ultrahigh magnetic field FT-ICR MS equipped with harmonized ICR cells.     

Studies of histidine as a suitable isoelectric buffer for tryptic digestion and isoelectric trapping fractionation followed by capillary electrophoresis-mass spectrometry for proteomic analysis

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.     

Alkylating tryptic peptides to enhance electrospray ionization mass spectrometry analysis

A major limitation of mass spectrometry-based proteomics is inefficient and differential ionization during electrospray ionization (ESI). This leads to problems such as increased limits of detection and incomplete sequence coverage of proteins. Incomplete sequence coverage is especially problematic for analyses that require the detection and identification of specific peptides from a protein, such as the analysis of post-translational modifications. We describe here the development and use of aldehyde-based chemistry for the alkylation of peptide primary amines to increase peptide hydrophobicity, providing increased ionization efficiency and concomitant signal enhancement. When employed to modify the peptide products of protein tryptic digests, increased sequence coverage is obtained from combined modified and unmodified digests. To evaluate the utility of alkylation of peptides for selected reaction monitoring (SRM) assays, we alkylated a peptide from the protein Oct4, known to play a role in regulating stem cell differentiation. Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form.     

High-sensitivity electron capture dissociation tandem FTICR mass spectrometry of microelectrosprayed peptides

Electron capture dissociation (ECD) has previously been shown by other research groups to result in greater peptide sequence coverage than other ion dissociation techniques and to localize labile posttranslational modifications. Here, ECD has been achieved for 10-13-mer peptides microelectrosprayed from 10 nM (10 fmol/microL) solutions and for tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein. Tandem Fourier transform ion cyclotron resonance (FTICR) mass spectra contain fragment ions corresponding to cleavages at all possible peptide backbone amine bonds, except on the N-terminal side of proline, for substance P and neurotensin. For luteinizing hormone-releasing hormone, all but two expected backbone amine bond cleavages are observed. The tandem FTICR mass spectra of the tryptic peptides contain fragment ions corresponding to cleavages at 6 of 12 (1545.7-Da peptide) and 8 of 21 (2944.5-Da peptide) expected backbone amine bonds. The present sensitivity is 200-2000 times higher than previously reported. These results show promise for ECD as a tool to produce sequence tags for identification of peptides in complex mixtures available only in limited amounts, as in proteomics.     

In situ liquid-liquid extraction as a sample preparation method for matrix-assisted laser desorption/ionization MS analysis of polypeptide mixtures

A novel liquid-liquid extraction (LLE) procedure was investigated for preparation of peptide and protein samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). LLE using ethyl acetate as the water-immiscible organic solvent enabled segregation of hydrophobic and hydrophilic polypeptides in mixtures, thereby reducing the complexity of mass spectra obtained by MALDI MS. The LLE technique was optimized for rapid and sensitive in situ (on-target) sample preparation for MALDI MS analysis of proteins and peptides at low-picomole and subpicomole levels. Addition of MALDI matrix to the organic solvent enhanced the efficiency of the LLE-MALDI MS method for analysis of hydrophobic peptides and proteins. LLE-MALDI MS enabled the detection of the hydrophobic membrane protein bacteriorhodopsin as a component in a simple protein mixture. Peptide mixtures containing phosphorylated, glycosylated, or acylated peptides were successfully separated and analyzed by the in situ LLE-MALDI MS technique and demonstrate the potential of this method for enhanced separation and structural analysis of posttranslationally modified peptides in proteomics research.     

Epitope Imprinting Technology: Progress,Applications, and Perspectives toward Artificial Antibodies

Epitope imprinting is a promising tool to generate antibody‐like specific recognition sites. Recently, because of the ease of obtaining templates, the flexibility in selecting monomers, their resistance to harsh environments, and the high specificity toward targets, epitope‐imprinted materials have attracted much attention in various fields, such as bioanalysis, clinical therapy, and pharmacy. Here, the discussion is focused on the current representative epitope imprinting technologies, including epitope bulk imprinting and epitope surface imprinting. Moreover, the application of epitope‐imprinted materials to the recognition of peptides, proteins, and cells is reviewed. Finally, the remaining challenges arising from the intrinsic properties of epitope imprinting are discussed, and future development in the field is prospected.     

Molecularly Imprinted Polymers as Synthetic Antibodies for Protein Recognition: The Next Generation

Molecularly imprinted polymers (MIPs) are chemical antibody mimics obtained by nanomoulding the 3D shape and chemical functionalities of a desired target in a synthetic polymer. Consequently, they possess exquisite molecular recognition cavities for binding the target molecule, often with specificity and affinity similar to those of antigen-antibody interactions. Research on MIPs targeting proteins began in the mid-90s, and this review will evaluate the progress made till now, starting from their synthesis in a monolith bulk format through surface imprinting to biocompatible soluble nanogels prepared by solid-phase synthesis. MIPs in the latter format will be discussed more in detail because of their tremendous potential of replacing antibodies in the biomedical domain like in diagnostics and therapeutics, where the workforce of antibodies is concentrated. Emphasis is also put on the development of epitope imprinting, which consists of imprinting a short surface-exposed fragment of a protein, resulting in MIPs capable of selectively recognizing the whole macromolecule, amidst others in complex biological media, on cells or tissues. Thus selecting the ‘best’ peptide antigen is crucial and in this context a rational approach, inspired from that used to predict peptide immunogens for peptide antibodies, is described for its unambiguous identification.     

Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:10(5) and 1:10(6). In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of approximately 0.5 fmol/microL in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic.     

Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures.

Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PMM) can be generated for protein identification. However, insufficient peptide mass accuracy and protein sequence coverage limit the potential of the PMM approach for high-throughput, large-scale analysis of proteins. In our novel approach, nonlabile protons in particular amino acid residues were replaced with deuteriums to mass-tag proteins of the S. cerevisiae proteome in a sequence-specific manner. The resulting mass-tagged proteolytic peptides with characteristic mass-split patterns can be identified in the data search using constraints of both amino acid composition and mass-to-charge ratio. More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identify the parent proteins at modest mass accuracy and low sequence coverage. As a result, the specificity and accuracy of a PMM was greatly enhanced without the need for peptide sequencing or instrumental improvements to obtain increased mass accuracy. The power of PMM has been extended to the unambiguous identification of multiple proteins in a 1D SDS gel band including the identification of a membrane protein.     

Comprehensive comparison of collision induced dissociation and electron transfer dissociation

Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues. However, unlike collision induced dissociation (CID), which has been studied for decades, the intricacies of ETD-based fragmentation have not yet been firmly established or systematically addressed. In this analysis, we have systematically compared the CID and ETD fragmentation patterns for the large majority of the peptides that do not contain such labile modifications. Using a standard 48 protein mix, we were able to measure false-positive rates for the experiments and also assess a large number of peptides for a detailed comparison of CID and ETD fragmentation pattern. Analysis of approximately 19,000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in approximately 20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%. Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted. We also present a novel strategy for automatic validation of peptide assignments based on identification of a peptide by consecutive CID and ETD fragmentation in an alternating mode.     

MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.     

Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification

Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.     

Approach for determining protein ubiquitination sites by MALDI-TOF mass spectrometry

Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the epsilon-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining posttraslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic peptides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves.     

The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for mass finger-printing of peptide mixtures obtained after enzymatic ingel digestion of proteins separated by two-dimensional electrophoresis (2-DE). In the course of a proteome analysis of mycobacteria using mass spectrometric identification, it was found that 94% of the most intense MALDI-MS peaks denote peptides bearing arginine at the C-terminal end. The effect was demonstrated to be equally prominent using an equimolar mixture of the synthetic peptides known to be present in the tryptic digest of the mycobacterial 35 kDa antigen ("synthetic mass map"). In addition, several binary mixtures of synthetic peptides differing exclusively at the C terminus (Arg or Lys) were examined to rationalize the higher sensitivity toward arginine-containing peptides. The extent of the effect described depends on the matrix used and may facilitate a more reliable assignment of mass fingerprint data to protein sequences in databases.     

Comparison of electrokinetics-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of human salivary proteins

The foundation for saliva-based diagnostics is the development of a complete catalog of secreted proteins detectable in saliva. Besides protein complexity, the greatest bioanalytical challenge facing comprehensive analysis of saliva samples is related to the large variation of protein relative abundances including the presence of high-abundance proteins such as amylases, mucins, proline-rich proteins (PRPs), and secretory IgA complex. Among a number of electrokinetic separation techniques, transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) specifically targets trace amounts of proteins and thus reduces the range of relative protein abundances for providing unparallel advantages toward the identification of low-abundance proteins. By employing a CITP/CZE-based multidimensional separation platform coupled with electrospray ionization-tandem mass spectrometry (ESI-tandem MS), a total of 6112 fully tryptic peptides are sequenced at a 1% false discovery rate (FDR), leading to the identification of 1479 distinct human SwissProt protein entries. By comparing with capillary isoelectric focusing (CIEF) as another electrokinetics-based stacking approach, CITP/CZE not only offers a broad field of application but also is less prone to protein/peptide precipitation during the analysis. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. Furthermore, when evaluating the protein sequence coverage by the number of distinct peptides mapping to each protein identification, the CITP-based proteome technology similarly achieves the superior performance with 674 proteins (46%) having three or more distinct peptides, 288 (19%) having two distinct peptides, and 517 (35%) having a single distinct peptide.