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This review considers the issues of molecular imprinting with the participation of proteins. Works in the field of bioimprinting published over the past five years and devoted to the determination of biomolecules and the enhancement of enzymatic activity are analyzed. Attention is focused on the imprinting of proteins as a method of modifying the molecular structure of proteins due to the formation of binding sites in the presence of substrates (proteins with molecular imprints or imprinted proteins). It is shown that the imprinting of proteins is promising for solving analytical problems. The ambiguous interpretation of the term bioimprinting in solving various problems is discussed., Author(s): P. S. Pidenko [sup.1] , K. Yu. Presnyakov [sup.1] , N. A. Burmistrova [sup.1] Author Affiliations: (1) grid.446088.6, 0000 0001 2179 0417, Institute of Chemistry, Saratov State University, , [...]

Epigenetic regulation contributes to human health and disease, especially cancer, but the mechanisms of many epigenetic regulators remain obscure. Most research is focused on gene regulatory processes, such as mRNA translation and DNA damage repair, rather than the effects on biological functions like mitochondrial activity and oxidative phosphorylation. Here, we identified an essential role for the histone chaperone structure-specific recognition protein 1 (SSRP1) in mitochondrial oxidative respiration in hepatocellular carcinoma, and found that SSRP1 suppression led to mitochondrial damage and decreased oxidative respiration. Further, we focused on TNF receptor-associated protein 1 (TRAP1), the only member of the heat shock protein 90 (HSP90) family which directly interacts with selected respiratory complexes and affects their stability and activity. We confirmed that SSRP1 downregulation caused a decrease in TRAP1 expression at both the mRNA and protein levels. A chromatin immunoprecipitation assay also showed that SSRP1 could deposit in the TRAP1 promoter region, indicating that SSRP1 maintains mitochondrial function and reactive oxygen species levels through TRAP1. Additionally rescue experiments and animal experiments confirmed the mechanism of SSRP1 and TRAP1 interaction. In summary, we identified a new mechanism that connects mitochondrial respiration and apoptosis, via SSRP1. Key words: epigenetics, SSRP1, TRAP1, mitochondrial respiration, ROS, hepatocellular carcinoma, Introduction Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers and is the second most fatal cancer worldwide (Lv et al. 2021). The initiation and progression of HCC [...]

Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported.sup.1, advances in mass-spectrometry-based proteomics.sup.2 have enabled increasingly comprehensive identification and quantification of the human proteome.sup.3-6. However, there have been few comparisons across species.sup.7,8, in stark contrast with genomics initiatives.sup.9. Here we use an advanced proteomics workflow--in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system--for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a large-scale case study for sequence-based machine learning, as we demonstrate by experimentally confirming the predicted properties of peptides from Bacteroides uniformis. Our results offer a comparative view of the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the biological challenge of maintaining protein structure in all branches of life. Likewise, a universally high fraction is involved in supplying energy resources, although these pathways range from photosynthesis through iron sulfur metabolism to carbohydrate metabolism. Generally, however, proteins and proteomes are remarkably diverse between organisms, and they can readily be explored and functionally compared at (http://www.proteomesoflife.org). An advanced proteomics workflow is used to identify 340,000 proteins from 100 taxonomically diverse species, providing a comparative view of proteomes across the evolutionary range., Author(s): Johannes B. Müller [sup.1] , Philipp E. Geyer [sup.1] [sup.2] , Ana R. Colaço [sup.3] , Peter V. Treit [sup.1] , Maximilian T. Strauss [sup.1] [sup.2] , Mario Oroshi [...]

The sympathetic nervous system drives brown and beige adipocyte thermogenesis through the release of noradrenaline from local axons. However, the molecular basis of higher levels of sympathetic innervation of thermogenic fat, compared to white fat, has remained unknown. Here we show that thermogenic adipocytes express a previously unknown, mammal-specific protein of the endoplasmic reticulum that we term calsyntenin 3[beta]. Genetic loss or gain of expression of calsyntenin 3[beta] in adipocytes reduces or enhances functional sympathetic innervation, respectively, in adipose tissue. Ablation of calsyntenin 3[beta] predisposes mice on a high-fat diet to obesity. Mechanistically, calsyntenin 3[beta] promotes endoplasmic-reticulum localization and secretion of S100b--a protein that lacks a signal peptide--from brown adipocytes. S100b stimulates neurite outgrowth from sympathetic neurons in vitro. A deficiency of S100b phenocopies deficiency of calsyntenin 3[beta], and forced expression of S100b in brown adipocytes rescues the defective sympathetic innervation that is caused by ablation of calsyntenin 3[beta]. Our data reveal a mammal-specific mechanism of communication between thermogenic adipocytes and sympathetic neurons. The newly identified calsyntenin 3[beta] protein has a role in the innervation of thermogenic fat through a mechanism of communication--which is unique to mammals--between thermogenic adipocytes and sympathetic neurons., Author(s): Xing Zeng [sup.1] [sup.2] , Mengchen Ye [sup.3] , Jon M. Resch [sup.4] , Mark P. Jedrychowski [sup.1] [sup.2] , Bo Hu [sup.1] [sup.2] , Bradford B. Lowell [sup.4] [...]

Silica colloidal crystals formed from 330 nm nonporous silica spheres inside of 75 [micro]m i.d. fused silica capillaries were evaluated for the efficiency of capillary electrochromatography of proteins. Three proteins, ribonuclease A, cytochrome C, and lysozyme, each covalently labeled with fluorophor, were well separated over a distance of 1 cm by isocratic electromigration, using 40:60 acetonitrile/ water with 0.1% formic acid. A van Deemter plot showed that the plate height for lysozyme, which was the purest of the three proteins, was diffusion-limited for electric fields ranging from 400 to 1400 V/cm. The plate height for lysozyme was below 50 nm at almost all of the migration velocities, and it approached 10 nm at the highest velocity. Eddy diffusion was negligible. Lysozyme migrated over a 12 mm separation length with more than [10.sup.6] plates in 1.5 min. These results indicate that silica colloidal crystals are well suited for electrically driven separations of large, highly charged analytes such as proteins. The [10.sup.6] plates observed for a separation length of barely more than a centimeter means they are potentially valuable for miniaturized separations in microchip and lab-on-a-chip devices. 10.1021/ac102438w

Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS2 strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein. 10.1021/ac1020722

Parallel sequence and structure alignment tools have become ubiquitous and invaluable at all levels in the study of biological systems. We demonstrate the application and utility of this same parallel search paradigm to the process of protein structure determination, benefitting from the large and growing corpus of known structures. Such searches were previously computationally intractable. Through the method of Wide Search Molecular Replacement, developed here, they can be completed in a few hours with the aide of national-scale federated cyberinfrastructure. By dramatically expanding the range of models considered for structure determination, we show that small (less than 12% structural coverage) and low sequence identity (less than 20% identity) template structures can be identified through multidimensional template scoring metrics and used for structure determination. Many new macromolecular complexes can benefit significantly from such a technique due to the lack of known homologous protein folds or sequences. We demonstrate the effectiveness of the method by determining the structure of a full-length p97 homologue from Trichoplusia ni. Example cases with the MHC/T-cell receptor complex and the EmoB protein provide systematic estimates of minimum sequence identity, structure coverage, and structural similarity required for this method to succeed. We describe how this structure-search approach and other novel computationally intensive workflows are made tractable through integration with the US national computational cyberinfrastructure, allowing, for example, rapid processing of the entire Structural Classification of Proteins protein fragment database. p97 ATPase | likelihood functions | scoring methods | grid computing doi/ 10.1073/pnas.1012095107

The major challenge of prion disease diagnosis at the presymptomatic stage is how to sensitively or selectively discriminate and detect the minute quantity of disease-associated prion protein isoform ([PrP.sup.Res]) in complex biological systems such as serum and brain homogenate. In this contribution, we developed a dual-aptamer strategy by taking the advantages of aptamers, the excellent separation ability of magnetic microparticles (MMPs), and the high fluorescence emission features of quantum dots (QDs). Two aptamers (Apt1 and Apt2), which can recognize their two corresponding distinct epitopes of prion proteins (PrP), were coupled to the surfaces of MMPs and QDs, respectively, to make MMPs-Aptl and QDs-Apt2 ready at first, which then could be coassociated together through the specific recognitions of the two aptamers with their two corresponding distinct epitopes of PrP, forming a sandwich structure of MMPs-Apt1-PrP-Apt2-QDs and displaying the strong fluorescence of QDs. Owing to the different binding affinities of the two aptamers with [PrP.sup.Res] and cellular prion protein ([PrP.sup.C]), both of which have distinct denaturing detergent resistance, our dual-aptamer strategy could be applied to discriminate [PrP.sup.Res] and [PrP.sup.C] successfully in serum. Further identifications showed that the present dual-aptamer assay could be successfully applied to the detection of PrP in 0.01% brain homogenate, about 1000-fold lower than that of commonly applied antibody-mediated assays, which can detect PrP just in 10% brain homogenate, indicating that the present designed dual-aptamer assay is highly sensitive and adequate for clinical diagnosis without isolation of target protein prior to assay. 10.1021/ac101865s

The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP 16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP 16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments. 10.1021/ac101778e

We report a new and label-free method to detect and characterize the clustering of membrane-bound proteins and, by extension, the lateral segregation of nanosized particles adsorbed to planar surfaces in liquid environment. The method exploits the contrast between two different mass and surface sensitive detection methods, quartz crystal microbalance with dissipation monitoring and ellipsometry. The time-resolved correlation of both techniques provides insight into subtle changes in the clustering state of surface-bound molecules that is not accessible with either technique alone. A theoretical model can provide quantitative predictions about the size of surface-bound clusters. 10.1021/ac102495q

Protein radicals are implicated in oxidative stress and are associated with a wide range of diseases and disorders. In the present work, we describe the specific application of a newly synthesized nitrone spin trap, Bio-SS-DMPO, for the detection of these highly reactive species by mass spectrometry (MS). Bio-SS-DMPO is a biotinylated analogue of the spin-trap 5,5-dimethyi-1-pyrroline-N-oxide (DMPO) that allows for specific capture of the protein(s)/peptide(s) labeled by the spin-trap on a (strept)avidin-bound solid matrix. The disulfide bond in the linker arm joining biotin to DMPO can be cleaved to release captured spin-adduct peptide from the solid matrix. This (strept)avidin-based affinity purification reduces the complexity of the samples prior to MS analyses, thereby facilitating the location of the sites of spin trap addition. In addition, the biotin moiety on the spin-trap can efficiently be probed with (strept)avidin-conjugated reporter. This offers an effective means to visualize the presence of DMPO-adducted proteins in intact cells. 10.1021/ac1023183

The HIV-1 capsid protein, p24 antigen, is of considerable diagnostic interest because following HIV exposure it is detectable several days earlier than host-generated HIV antibodies (which are the target of almost all current tests used in the field) and can be used to design very sensitive assays without the need for PCR. Here, we present an ultrasensitive capacitive immunosensor that is capable of detecting subattagram per milliliter concentrations of p24 antigen, which to our knowledge is the lowest level of detection ever reported. Dilution studies using p24-spiked human plasma samples indicate that the immunosensor is robust against the interfering effects of a complex biological matrix. Moreover, the capacitive immunosensor assay is rapid ( 10.1021/ac102144a

Proteins are one of the most versatile groups of molecules with vital functional roles in living systems. Their enormous diversity and structural flexibility make the detection of these molecules a challenging task. A simple and sensitive label-free protein detection method based on assembly of proteins and colloidal silver nanoparticles (AgNPs) on surfaces and surface-enhanced Raman scattering (SERS) is reported. The SERS spectra from the assembled AgNP/protein films show excellent reproducibility and high quality regardless of the proteins' charge status and size. A detection limit down to 0.5 [micro]g/mL for three acidic proteins; BSA, catalase and pepsin, and three basic proteins; cytochrome c, avidin and lysozyme, is easily achieved. The minimum improvement in detection limit is more than 1 order of magnitude compared to the previously reported detection limits using the technique and the approach has the potential for label-free protein detection and identification. 10.1021/ac101720s

Among the hyphenated techniques used to probe and identify metalloproteins, size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS) has shown to have a central place. However, the calibration of SEC columns reveals to be tedious and always involves UV detection prior to ICP-MS. The presence of sulfur in 98% of proteins allows their detection by quadrupole ICP-MS, despite the isobaric interference ([sup.16]O[sup.16]O) on S, by monitoring [sup.32]S[sup.16]O at mass to charge ratio (m/z) 48. The formation of SO occurs spontaneously in the argon plasma but can be optimized by the introduction of oxygen gas into a reaction cell (RC) to achieve nM levels. In this article, sulfur detection was discussed upon instrumental conditions and S detection was then optimized by applying O2 as a reaction gas. SO formation was used to calibrate SEC columns without IN detection. This simple SEC-ICPMS method was used for plasma copper proteins in plasma healthy subjects (HS) and an untreated Wilson disease (WD) patient. Copper proteins identified in healthy subjects were transcuprein, ceruloplasmin (Cp) and albumin. The method led to results in good agreement with other methods of determination. Copper bound to Cp in the WD patient was lowered with regard to the HS, and the exchangeable Cu was highly increased. 10.1021/ac101128x

A bacterial protein in disguise works to squelch an infected host's immune system. A bacterial protein in disguise works to squelch an infected host's immune system., Author Affiliations: A leaf blighted by the bacterium Pseudomonas syringae , which deploys a protein that mimics those of the plants it infects. [see PDF for image] Credit: David J. [...]

We have analyzed peptides, proteins, and protein--drug complexes through surface-assisted laser desorption/ ionization mass spectrometry (SALDI-MS) using HgTe nanostructures as matrixes. We investigated the effects of several parameters, including the concentration of the HgTe nanostructures, the pH of the buffer, and the concentration of salt, on the performance of this system. When HgTe nanostructures are used as matrixes, [[M + H].sup.+] ions were the dominant signals. Relative to other commonly used nanomaterials, HgTe nanostructures provided lower background signals from metal clusters, fewer fragment ions, less interference from alkali-adducted analyte ions, and a higher mass range (up to 150 000 Da). The present approach provides limits of detection for angiotensin I and bovine serum albumin of 200 pM and 14 nM, respectively, with great reproducibility (RSD: 10.1021/ac100550c

Coomassie brilliant dyes have high affinity to proteins and high Raman activity, and on the basis of which, we have employed brilliant blue R-250 (BBR) and brilliant blue G-250 (BBG) as surface-enhanced Raman scattering (SERS) labels to probe protein--ligand recognitions. This method differs from previously proposed methods in that target proteins are labeled rapidly before biological recognitions without procedures of separation and purification, rather than attaching Raman labels to metal nanoparticles, which significantly simplifies the Raman dye labeling procedure. In typical assays, ligand-functionalized metal nanoparticles assemble by target protein-specific bindings and this assembly sequentially turns on electromagnetic enhancement of Raman scattering of the proposed labels. The method with its advantages of rapidness, high sensitivity, and spectral multiplexing has great potential in probing protein protein and protein small molecule recognitions not only in solution systems but also on flexible solid substrates. 10.1021/ac100202w

Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is a powerful technique giving access to the distribution of a large range of biomolecules directly from a tissue section, allowing, for example, the discovery of new pathological biomarkers. Nevertheless, one main difficulty lies in the identification of the detected species, especially proteins. MALDI-in source decay (ISD) is used to fragment ions directly in the mass spectrometer ion source. This technique does not require any special sample treatment but only the use of a specific MALDI matrix such as 2,5-dihydroxybenzoic acid or 1,5-diaminonaphthalene. MALDI-ISD is generally employed on classical, purified samples, but here we demonstrate that ISD can also be performed directly on mixtures and on a tissue slice leading to fragment ions, allowing the identification of major proteins without any further treatment. On a porcine eye lens slice, de novo sequencing was even performed. Crystallins not yet referenced in databases were identified by sequence homology with other mammalian species. On a mouse brain slice, we demonstrate that results obtained with ISD are comparable and even better than those obtained with a classical in situ digestion. 10.1021/ac902875q

Proteins mediate the bulk of biological activity and are powerfully assayed in the diagnosis of diseases. Protein detection relies largely on antibodies, which have significant technical limitations especially when immobilized on two-dimensional surfaces. Here, we report the integration of peptide aptamers with extended gate metal-oxide-semiconductor field-effect transistors (MOSFETs) to achieve label-free sub-picomolar target protein detection. Specifically, peptide aptamers that recognize highly related protein partners of the cyclin-dependent kinase (CDK) family are immobilized on the transistor gate to enable human CDK2 to be detected at 100 fM or 5 pg/ mL, well within the clinically relevant range. The target specificity, ease of fabrication, and scalability of these FET arrays further demonstrate the potential application of the multiplexable field effect format to protein sensing. 10.1021/ac902554v

A cascade signal amplification strategy was proposed for detection of protein target at ultralow concentration by combining the rolling circle amplification (RCA) technique with oligonucleotide functionalized quantum dots (QDs), multiplex binding of the biotin--strepavidin system, and anodic stripping voltammetric detection. The RCA product containing tandem-repeat sequences could serve as excellent template for periodic assembly of QDs, which presented per protein recognition event to numerous quantum dot tags for electrochemical readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human vascular endothelial growth factor as a model protein, the designed strategy could quantitatively detect protein down to 16 molecules in a 100 [micro]L sample with a linear calibration range from 1 aM to 1 pM and was amenable to quantification of protein target in complex biological matrixes. The proposed cascade signal amplification strategy would become a powerful tool for proteomics research and clinical diagnostics. 10.1021/ac100144g

Multidimensional separation is often applied for large-scale qualitative and quantitative proteome analysis. A fully automated system with integration of a reversed phase-strong cation exchange (RP-SCX) biphasic trap column into vented sample injection system was developed to realize online sample loading, isotope dimethyl labeling and online multidimensional separation of the proteome samples. Comparing to conventionally manual isotope labeling and off-line fractionation technologies, this system is fully automated and time-saving, which is benefit for improving the quantification reproducibility and accuracy. As phosphate SCX monolith was integrated into the biphasic trap column, high sample injection flow rate and high-resolution stepwise fractionation could be easily achieved. ~ 1000 proteins could be quantified in ~30 h proteome analysis, and the proteome coverage of quantitative analysis can be further greatly improved by prolong the multidimensional separation time. This system was applied to analyze the different protein expression level of HCC and normal human liver tissues. After three times replicated analysis, finally 94 up-regulated and 249 down-regulated (HCC/Normal) proteins were successfully obtained. These significantly regulated proteins are widely validated by both gene and proteins expression studies previously. Such as some enzymes involved in urea cycle, methylation cycle and fatty acids catabolism in liver were all observed down-regulated. 10.1021/ac100075y

Many cellular processes are driven by protein complexes. Although the identification of protein components in such complexes has become almost a routine matter, accurate determination of their stoichiometry within a protein complex is still a challenge. We have established a method to determine the stoichiometries of protein complexes using absolute quantification (AQUA) with the help of synthetic standard peptides in combination with multiple reaction monitoring (MRM). Our approach is exemplified by the analysis of the human spliceosomal hPrpl9/ CDC5L complex, which consists of seven individual proteins and plays a crucial role in the assembly of the fully catalytically active spliceosome during pre-mRNA splicing. We evaluated several conditions for complete hydrolysis of the protein complex and found that the denaturing conditions under which hydrolysis is performed arc absolutely crucial for accurately determining protein stoichiometries within this complex. In addition, we tested the suitability of different AQUA peptides and further compared different MS techniques to read out the relative signal intensities that were then used in absolute quantification. Our analyses revealed that dependent on the denaturing conditions different stoichiometries within the complex were obtained. The most consistent results were obtained by enzymatic hydrolysis in the presence of acetonitrile in combination with MRM. 10.1021/ac902710k

Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight time-of-flight tandem mass spectrometry (MALD1-TOF--TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALD1, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli Ol57:H7 and 7 strains of the 'near-neighbor' serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, non-pathogenic E. coli by MALDI-TOF--TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z ~9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics. 10.1021/ac902455d

Liquid chromatography (LC)/mass spectrometry (MS) in selected-reactions-monitoring (SRM) mode provides a powerful tool for targeted protein quantification. However, efficient, high-throughput strategies for proper selection of signature peptides (SP) for protein quantification and accurate optimization of their SRM conditions remain elusive. Here we describe an on-the-fly, orthogonal array optimization (OAO) approach that enables rapid, comprehensive, and reproducible SRM optimization of a large number of candidate peptides in a single nanoflow-LC/MS run. With the optimized conditions, many peptide candidates can be evaluated in biological matrixes for selection of the final SP. The OAO strategy employs a systematic experimental design that strategically varies product ions, declustering energy, and collision energy in a cycle of 25 consecutive SRM trials, which accurately reveals the effects of these factors on the signal-to-noise ratio of a candidate peptide and optimizes each. As proof of concept, we developed a highly sensitive, accurate, and reproducible method for the quantification of carbonyl reductases CBR1 and CBR3 in human liver. Candidate peptides were identified by nano-LC/LTQ/Orbitrap, filtered using a stringent set of criteria, and subjected to OAO. After evaluating both sensitivity and stability of the candidates, two SP were selected for quantification of each protein. As a result of the accurate OAO of assay conditions, sensitivities of 80 and 110 amol were achieved for CBR1 and CBR3, respectively. The method was validated and used to quantify the CBRs in 33 human liver samples. The mean level of CBR1 was 93.4 [+ or -] 49.7 (range: 26.2-241) ppm of total protein, and of CBR3 was 7.69 [+ or -] 4.38 (range: 1.26-17.9) ppm. Key observations of this study: (i) evaluation of peptide stability in the target matrix is essential for final selection of the SP; (ii) utilization of two unique SP contributes to high reliability of target protein quantification; (iii) it is beneficial to construct calibration curves using standard proteins of verified concentrations to avoid severe biases that may result if synthesized peptides alone are used. Overall, the OAO method is versatile and adaptable to high-throughput quantification of validated biomarkers identified by proteomic discovery experiments. 10.1021/ac902314m

A reusable aptameric recognition system was described for the electrochemical detection of the protein PDGF-BB based on the target binding-induced rolling circle amplification (RCA). A complementary DNA (CDNA), linear padlock probe, and primer probe were utilized to introduce a RCA process into the aptamer--target binding event while a new aptamer was elegantly designed via lengthening the original aptamer by the complement to the CDNA. The aptameric sensing system facilitates the integration of multiple functional elements into a signaling scheme: a unique electrochemical technique, an attractive RCA process, reversible DNA hybridization, and desirable aptameric target recognition. This RCA-based electrochemical recognition system not only exhibits excellent performance (e.g., a detection limit of 6.3 x [10.sup.-11] M, a linear dynamic range of 2 orders of magnitude, high specificity, and satisfactory repeatability) but also overcomes the limitations associated with conventional aptameric biosensors (e.g., dependence of signaling target binding on specific aptamer sequence or requirement of sandwich assays for two or more binding sites per target molecule). A recovery test demonstrated the feasibility of the developed target protein assay. Given the attractive characteristics, this aptameric recognition platform is expected to be a candidate for the detection of proteins and other ligands of interest in both fundamental and applied research. 10.1021/ac902400n

A universal approach is proposed in this study for the development of an aptameric assay system for proteins based on aptamer structure-switching-triggered ligation-rolling circle amplification (L-RCA) upon target binding. The strategy chiefly depends on the competition for binding the aptamer probe between target protein and a complementary single-stranded DNA (CDNA) that can induce the circularization of the padlock probe. Introduction of target protein into the assay system inhibits the hybridization of the CDNA with the aptamer probe because of the formation of the target/aptamer duplex. The free CDNA can only hybridize with the padlock probe. With the assistance of DNA ligase, the padlock probe is circularized, and the subsequent RCA process can be accomplished by Phi 29 DNA polymerase. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons (detection probes), resulting in an enhanced fluorescence signal. In contrast, in the absence of target protein, no obvious change in the fluorescence intensity of the detection probe is observed. This signaling mode for target recognition and transduction events is based on the combination of aptamer recognition elements and L-RCA technology with high specificity and sensitivity. The proposed assay system not only exhibits excellent analytical characteristics (e.g., the detection limit on attomolar scale and a linear dynamic range of more than 3 orders of magnitude) but also possesses significant advantages over existing aptameric assays. The proposed strategy is universal since the sequences of aptamer probe, CDNA, and padlock probe could be easily designed to be compatible with the L-RCA based detection of other proteins without other conditions. 10.1021/ac901794w

The application of the direct electrospray ionization mass spectrometry (ES-MS) assay for quantifying the stoichiometry and absolute affinity of protein--metal ion binding in vitro is described. Control ES-MS experiments performed on solutions containing calcium chloride or calcium acetate and a pair of proteins that do not bind calcium ions in solution revealed that the nonspecific association of metal ions to proteins during ES is a random process, independent of protein size and structure. These results establish the reliability of the reference protein method for quantitatively correcting ES mass spectra for the occurrence of nonspecific metal ion binding to proteins during ES-MS analysis. To demonstrate the utility of the direct ES-MS assay, when carried out using the reference protein method, the calcium binding stoichiometry of bovine [alpha]-lactalbumin and the calcium ion affinity of bovine [beta]-lactoglobulin were established. 10.1021/ac902633d

Liquid chromatography, coupled with tandem mass spectrometry, is an established method for the identification of proteins from a complex sample. Despite its wide application, the analysis of whole proteomes still represents a challenge to researchers, because of the complexity and dynamic range of protein concentrations in biological samples. The analysis of such samples can be improved by adding a prefractionation step or a combination of orthogonal separation techniques. Off-gel isoelectric focusing (OGE) has successfully been used for prefractionation of a tryptic digest prior to nLC separation. In contrast to previous published results, we present a complete giycerol-free OGE for the analysis of purple membranes and Corynebacterium glutamicum membranes using the less-specific enzyme elastase. More than 85% of the identified unique peptides were found in solely one fraction, with very little carryover. These results are in accordance with those published for tryptic peptides. Therefore, OGE can be used as an effective prefractionation method in a multidimensional separation experiment of nontryptic membrane peptides. 10.1021/ac902776h

Nonspecific protein adsorption on self-assembled monolayer (SAM) alkanethiols with various terminal groups was investigated qualitatively and quantitatively using an extended gate-field effect transistor (extended gate-FET). The SAMs were characterized by XPS, cyclic voltammogram and water contact angle measurements. Changes in gate voltage of 1 mV caused by intrinsic charges of adsorbed protein on an undecanethiol SAM in 15 mM Dulbecco's phosphate buffered saline were equivalent to 3.6 ng [cm.sup.-2], 1.3 ng [cm.sup.-2], and 16 ng [cm.sup.-2] for bovine serum albumin (BSA), lysozyme, and bovine plasma fibrinogen (BPF), respectively, as calculated by the Debye Huckel model. Adsorption coefficients, maximum adsorption densities, and Gibbs free energies of adsorption were successfully determined using the Langmuir equation. The isotherms depended on the surface properties of the SAMs for BSA and lysozyme adsorption. In contrast, changes in gate voltage were almost independent of SAM type for BPF adsorption. Adsorption of large proteins may not be quantitatively detected because of the large dimensions of the biomolecules compared with the Debye length. In summary, the FET measurement is a nonlabellng, highly sensitive, and quantitative method for detecting nonspecific adsorption of small proteins with dimensions that are comparable to the Debye length of a solution. 10.1021/ac902401y

The methylmercury ion (C[H.sub.3][Hg.sup.+]) demonstrated a high efficiency for directly labeling peptide/protein based on its specific and strong interaction with the sulfhydryl(s) in the peptide/protein and because of its smallest size among monofunctional organic mercurials studied, including methylmercury, ethylmercury, 4-(hydroxymercuric)benzoic acid, and 2,7-dibromo-4-hydroxymercurifluoresceine disodium. A simple 1:1 stoichiometry between C[H.sub.3][Hg.sup.+] and sulthydryl, confirmed with electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOFMS) studies, made it easy to calibrate the stoichiometry of Hg in the peptide/protein. In order to avoid the direct use of the harmful C[H.sub.3][Hg.sup.+], in this study a C[H.sub.3][Hg.sup.+]-equivalent tag, methylmercurithiosalicylate (C[H.sub.3]Hg-THI), and its [sup.204]Hg-enriched homologue (C[H.sub.3][sup.204]Hg-THI) were synthesized, and then C[H.sub.3][Hg.sup.+] and/or C[H.sub.3][sup.204][Hg.sup.+] released from C[H.sub.3]Hg-THI and/or C[H.sub.3]Hg-THI in solution were utilized to demonstrate the dynamic labeling of glutathione (GSH) and two model proteins, [beta]-lactoglobulin (BLG) and ovalbumin (OVA), for the first time. Furthermore, the C[H.sub.3][sup.204]HgTHI isotopical labeled GSH, BLG, and OVA standards (C[H.sub.3][sup.204]Hg-GSH, C[H.sub.3][sup.204]Hg-BLG, and C[H.sub.3][sup.204]Hg-OVA) were used to demonstrate the feasibility of absolute peptide/protein quantification using label-specific isotope dilution inductively coupled plasma mass spectrometry (ICPMS). On the basis of the accurate and sensitive determination of Hg using ICPMS, the detection limits of GSH, BLG, and OVA could reach 45.4, 45.4, and 15.1 pmol [L.sup.-1], respectively, suggesting the possibility for low-abundance peptide/protein quantification alongside the surefire quantification of moderate and highly abundant peptide/protein. 10.1021/ac902902y

Fast identification of protein structures that are similar to a specified query structure in the entire Protein Data Bank (PDB) is fundamental in structure and function prediction. We present FragBag: An ultrafast and accurate method for comparing protein structures. We describe a protein structure by the collection of its overlapping short contiguous backbone segments, and discretize this set using a library of fragments. Then, we succinctly represent the protein as a 'bags-of-fragments'--a vector that counts the number of occurrences of each fragment--and measure the similarity between two structures by the similarity between their vectors. Our representation has two additional benefits: (i) it can be used to construct an inverted index, for implementing a fast structural search engine of the entire PDB, and (ii) one can specify a structure as a collection of substructures, without combining them into a single structure; this is valuable for structure prediction, when there are reliable predictions only of parts of the protein. We use receiver operating characteristic curve analysis to quantify the success of FragBag in identifying neighbor candidate sets in a dataset of over 2,900 structures. The gold standard is the set of neighbors found by six state of the art structural aligners. Our best FragBag library finds more accurate candidate sets than the three other filter methods: The SGM, PRIDE, and a method by Zotenko et al. More interestingly, FragBag performs on a par with the computationally expensive, yet highly trusted structural aligners STRUCTAL and CE. evaluation of structure search | fast structural search of Protein Data Bank | filter and refine | protein backbone fragments | protein structure search doi/10.1073/pnas.0914097107

The amylome is the universe of proteins that are capable of forming amyloid-like fibrils. Here we investigate the factors that enable a protein to belong to the amylome. A major factor is the presence in the protein of a segment that can form a tightly complementary interface with an identical segment, which permits the formation of a steric zipper--two self-complementary beta sheets that form the spine of an amyloid fibril. Another factor is sufficient conformational freedom of the self-complementary segment to interact with other molecules. Using RNase A as a model system, we validate our fibrillogenic predictions by the 3D profile method based on the crystal structure of NNQQNY and demonstrate that a specific residue order is required for fiber formation. Our genome-wide analysis revealed that self-complementary segments are found in almost all proteins, yet not all proteins form amyloids. The implication is that chaperoning effects have evolved to constrain self-complementary segments from interaction with each other. 3D profile | ribonuclease A | Rosetta energy | steric zipper doi/10.1073/pnas.0915166107

Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-to-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without nuclear magnetic resonance (NMR) information on the side chains for proteins up to 25 kilodaltons by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modeling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search toward the lowest-energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all-atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kilodaltons and should enable routine NMR structure determination for larger proteins. 10.1126/science.1183649

Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing bioanalytical and biotechnologlcal devices. Here, we report an approach for efficient site-specific protein immobilization based on enzymatic phosphopantetheinyi transfer (PPT) from coenzyme A (CoA)-functionalized glass-type surfaces to specific peptide tags. We devised a bottom-up surface modification approach for coupling CoA densely to a molecular poly(ethylene glycol) polymer brush. Site-specific enzymatic immobilization of proteins fused to different target peptides for the PPTase Sfp was confirmed by real-time label-free detection. Quantitative protein-protein interaction experiments confirmed that significantly more than 50% of the immobilized protein was fully active. The method was successfully applied with different proteins. However, different immobilization efficiencies of PPT-based immobilization were observed for different peptide tags being fused to the N- and C-termini of proteins. On the basis of this immobilization method, we established photolithographic patterning of proteins into functional binary microstructures. 10.1021/ac902608a

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 dimethyerylate) 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. 10.1021/ac9025362

Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins > 50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle skimmer dissociation and detection of fragment ions with 10.1021/ac9021083

Isoelectric focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomies. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly(vinyl alcohol) (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370 V/cm), efficient cooling maintains solution temperature inside the separation channel controllably in the range 2-25[degrees]C. Protein samples with a dynamic concentration range from pp/mL to mp/mL can be loaded into the microdevice at a flow rate of 1 mL/h and residence time of ~12 min. By using a protein complex of nine proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device-to-device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10-20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonsWated as an example. The microdevice has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches. 10.1021/ac902157e

Thermally addressed immunoassay is developed to detect multiple proteins using phase change nanoparticles as thermal barcodes. The solid to liquid phase changes of nanoparticles absorb heat energy and generate sharp melting peaks, which are used as thermal signatures to determine the existence and concentration of proteins. Multiple proteins can be detected by using different types of nanoparticles in order to create a one-to-one correspondence between one type of nanoparticle and one type of protein. The fusion enthalpy that is proportional to the amount of phase change materials has been used to derive the amount of protein. The melting temperatures of nanoparticles are designed to be higher than 100[degrees]C to avoid interference from species contained in the fluid. Thus, the use of thermal nanoparticles allows the detection of multiple low concentration proteins in a complex fluid such as cell lysate regardless of the color, salt concentration, and conductivity of the sample. 10.1021/ac902724y

The rapid and complete digestion of preteins is important when protein characterization by hydrogen-deuterium exchange (HDX) is coupled with mass spectrometry. We developed a single-pump, online, high-pressure digestion system that relies on UPLC technology to aid in the digestion of proteins. Two model proteins, amyloid [beta]-peptide 1-42 (A[beta] 1-42) and an HIV-1 capsid mutant protein (NBSA), were used to demonstrate the efficacy of the high-pressure system. Both model proteins readily aggregate and are difficult to digest under normal conditions. Our high-pressure system successfully digests these proteins into small, overlapping peptides. The extra information afforded by overlapping peptides allows us to pinpoint HDX protection to protein segments smaller than the digested peptide. The calculated average segment length (ASL) for both model proteins decreased by 2-fold for high-pressure digestion compared to digestion at ambient pressure. 10.1021/ac902477u

Multiply deprotonated anions [[M - nH].sup.n-] of large peptide mellitin, ubiquitin, and [beta]-casein proteins were subjected to laser irradiation at 260 nm in a quadrupole ion trap. For all compounds, the predominant event consecutive to laser irradiation was the detachment of an electron. The subsequent isolation and collisional activation of the oxidized [[M - nil].sup.(n-1)-*] resulted in extensive fragmentation of the peptide backbone. For mellitin peptide, nearly a complete series of c*, z, and a*, x product ions were observed. Applied to proteins, this technique, coined as activated-electron photodetachment dissociation (activated-EPD), achieved much more extensive sequence coverage than regular collision activated dissociation (CAD) on the even-electron components. Furthermore, the activated-EPD spectrum of [beta]-casein displayed phosphorylated fragment ions which suggest that the method is able to preserve part of the labile bonds of post-translational modifications. Activated-EPD is, therefore, a promising complementary technique to other dissociation techniques governed by radicals, i.e., electron capture dissociation (ECD), electron transfer dissociation (ETD), and electron detachment dissociation (EDD), for the structural characterization of large peptides and small proteins. 10.1021/ac901304d

Covalent modifications on therapeutic proteins are traditionally monitored by chromatographic techniques, which quantify limited number of protein modifications at a time. In this report, computer algorithms for automated analyses of liquid chromatography/tandem mass spectrometry (LC/MS/MS) data for large-scale identification and quantification of known and unknown modifications are described. Peptide identification is achieved by comparing the experimental fragmentation spectrum to the predicted spectrum of each native or modified peptide. Peak areas of related peptide ions under their selected-ion chromatograms (SIC) are used for relative quantification of modified peptides. A matched window function is used to generate SIC for more reliable quantification. In an LC/ MS/MS analysis of a tryptic digestion of an IgG2 monoclonal antibody, 1712 peptide ions were identified with a false-discovery rate of ~0.4%, and 227 modifications were identified and quantified. The accuracy of the mass spectrometry-based quantification is evaluated by comparing the abundance of different glycoforms determined by mass spectrometry to that determined by a fluorescence-based chromatography method. This large-scale method may potentially replace many chromatographic methods for assessing the quality attributes of therapeutic proteins. 10.1021/ac901193n

It has become increasingly apparent that geometric relationships often exist between regions of two proteins that have quite different global topologies or folds. In this article, we examine whether such relationships can be used to infer a functional connection between the two proteins in question. We find, by considering a number of examples involving metal and cation binding, sugar binding, and aromatic group binding, that geometrically similar protein fragments can share related functions, even if they have been classified as belonging to different folds and topologies. Thus, the use of classifications inevitably limits the number of functional inferences that can be obtained from the comparative analysis of protein structures. In contrast, the development of interactive computational tools that recognize the 'continuous' nature of protein structure/function space, by increasing the number of potentially meaningful relationships that are considered, may offer a dramatic enhancement in the ability to extract information from protein structure databases. We introduce the MarkUs server, that embodies this strategy and that is designed for a user interested in developing and validating specific functional hypotheses. protein fold space | protein function annotation | protein structure alignment | protein structure similarity doi/ 10.1073/pnas.0907971106

Identification of proteins in the mammalian growth cone has the potential to advance our understanding of this critical regulator of neuronal growth and formation of neural circuit; however, to date, only one growth cone marker protein, GAP-43, has been reported. Here, we successfully used a proteomic approach to identify 945 proteins present in developing rat forebrain growth cones, including highly abundant, membrane-associated and actin-associated proteins. Almost 100 of the proteins appear to be highly enriched in the growth cone, as determined by quantitative immunostaining, and for 17 proteins, the results of RNAi suggest a role in axon growth. Most of the proteins we identified have not previously been implicated in axon growth and thus their identification presents a significant step forward, providing marker proteins and candidate neuronal growth-associated proteins. GAP-43 | proteomics | RNAi | neuronal growth-associated proteins | axon guidance

A thermosensitive macroporous hydrogel showing selectivity for the lysozyme was developed by an imprinting procedure that is based on metal coordinate interaction. A metal chelate monomer [N-(4-vinyl)-benzyl iminodiacetic acid] forming coordination complex with the template protein in the presence of Cu ions co-polymerized with N-isopropylacrylamide and acrylamide, using N,N-methylenebisactylamide as the cross-linker to prepare the thermosensitive protein-imprinted hydrogel. The synergetic combination of the smart property of the macroporous thermosensitive hydrogel with the merits of the coordinate interaction improved the selectivity and adsorption capacity, with respect to template lysozyme. The macropores were created by the frozen polymerization, and the influences of frozen polymerization and the chelate monomer content on the hydrogel affinity were investigated. The imprinted hydrogel can respond not only to external stimuli, but also to the template protein with a certain degree of shrinking. In recognition of the protein, the interaction of the imprinted thermosensitive hydrogel to the protein can be switched between the coordinate effect and the electrostatic effect by adding or not adding Cu ions. Finally, this imprinted hydrogel was used to purify the template lysozyme from the mixture of proteins and the real sample, which demonstrated its high selectivity.