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Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer’s safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvβ6., Knottin is a cystine knot peptide. Here, the authors develop a knottin-based tracer for positron emission tomography and demonstrate its ability to detect cancer and idiopathic pulmonary fibrosis through selective binding to integrin αvβ6.

Mass spectrometry imaging is a powerful tool for investigating the spatial distribution of chemical compounds in a biological sample such as tissue. Two common goals of these experiments are unsupervised segmentation of images into newly discovered homogeneous segments and supervised classification of images into predefined classes. In both cases, the important secondary goals are to characterize the uncertainty associated with the segmentation and with the classification and to characterize the spectral features that define each segment or class. Recent analysis methods have focused on the spatial structure of the data to improve results. However, they either do not address these secondary goals or do this with separate post hoc procedures. We introduce spatial shrunken centroids, a statistical model-based framework for both supervised classification and unsupervised segmentation. It takes as input sets of previously detected, aligned, quantified, and normalized spectral features and expresses both spatial and multivariate nature of the data using probabilistic modeling. It selects informative subsets of spectral features that define each unsupervised segment or supervised class and quantifies and visualizes the uncertainty in spatial segmentations and in tissue classification. In the unsupervised setting, it also guides the choice of an appropriate number of segments. We demonstrate the usefulness of this framework in a supervised human renal cell carcinoma experimental dataset and several unsupervised experimental datasets, including a pig fetus cross-section, three rodent brains, and a controlled image with known ground truth. This framework is available for use within the open-source R package Cardinal as part of a full pipeline for the processing, visualization, and statistical analysis of mass spectrometry imaging experiments.

High performance liquid chromatography (HPLC) is currently one of the most powerful analytical tools that has revolutionized the field of proteomics. Formerly known as high pressure liquid chromatography, this technique was introduced in the early 1960s to improve the efficiency of liquid chromatography separations using small stationary phase particles packed in columns. Since its introduction, continued advancements in column technology, development of different stationary phase materials and improved instrumentation has allowed the full potential of this technique to be realized. The various modes of HPLC in combination with mass spectrometry has evolved into the principal analytical technique in proteomics. It is now common practice to combine different types of HPLC in a multidimensional workflow to identify and quantify peptides and proteins with high sensitivity and resolution from limited amounts of samples. More recently, the introduction of Ultra High Performance Liquid Chromatography (UHPLC) has further raised the level of performance of this technique with significant increases in resolution, speed and sensitivity. The number of applications of HPLC and UHPLC in proteomics has been rapidly expanding and will continue to be a pivotal analytical technique. The aim of the following sections is to familiarize the beginner with the various HPLC methods routinely used in proteomics and provide sufficient practical knowledge regarding each of them to develop a separation and analytical protocol.

Cardinal is an R package for statistical analysis of mass spectrometry-based imaging (MSI) experiments of biological samples such as tissues. Cardinal supports both Matrix-Assisted Laser Desorption/Ionization (MALDI) and Desorption Electrospray Ionization-based MSI workflows, and experiments with multiple tissues and complex designs. The main analytical functionalities include (1) image segmentation, which partitions a tissue into regions of homogeneous chemical composition, selects the number of segments and the subset of informative ions, and characterizes the associated uncertainty and (2) image classification, which assigns locations on the tissue to pre-defined classes, selects the subset of informative ions, and estimates the resulting classification error by (cross-) validation. The statistical methods are based on mixture modeling and regularization. Contact: o.vitek@neu.edu Availability and implementation: The code, the documentation, and examples are available open-source at www.cardinalmsi.org under the Artistic-2.0 license. The package is available at www.bioconductor.org.

High performance liquid chromatography (HPLC) is currently one of the most powerful analytical tools that has revolutionized the field of proteomics. Formerly known as high pressure liquid chromatography, this technique was introduced in the early 1960s to improve the efficiency of liquid chromatography separations using small stationary phase particles packed in columns. Since its introduction, continued advancements in column technology, development of different stationary phase materials and improved instrumentation has allowed the full potential of this technique to be realized. The various modes of HPLC in combination with mass spectrometry has evolved into the principal analytical technique in proteomics. It is now common practice to combine different types of HPLC in a multidimensional workflow to identify and quantify peptides and proteins with high sensitivity and resolution from limited amounts of samples. More recently, the introduction of Ultra High Performance Liquid Chromatography (UHPLC) has further raised the level of performance of this technique with significant increases in resolution, speed and sensitivity. The number of applications of HPLC and UHPLC in proteomics has been rapidly expanding and will continue to be a pivotal analytical technique. The aim of the following sections is to familiarize the beginner with the various HPLC methods routinely used in proteomics and provide sufficient practical knowledge regarding each of them to develop a separation and analytical protocol.

MALDI mass spectrometry imaging (MSI) is emerging as a tool for protein and peptide imaging across tissue sections. Despite extensive study, there does not yet exist a baseline study evaluating the potential capabilities for this technique to detect diverse proteins in tissue sections. In this study, we developed a systematic approach for characterizing MALDI-MSI workflows in terms of limits of detection, coefficients of variation, spatial resolution, and the identification of endogenous tissue proteins. Our goal was to quantify these figures of merit for a number of different proteins and peptides, in order to gain more insight in the feasibility of protein biomarker discovery efforts using this technique. Control proteins and peptides were deposited in serial dilutions on thinly sectioned mouse xenograft tissue. Using our experimental setup, coefficients of variation were

A novel approach to immunoaffinity MS is described wherein antibodies are appended to a patterned gold Biochip surface. The Biochip surface is patterned with an array of concentric immunocapture zones composed of highly hydrophilic central zones surrounded by moderately hydrophilic zones that reside on a non-wetting background, with protein attachment via electrochemically cleavable linkers. After linker cleavage, matrix application forms a discrete spot suitable for MALDI-TOF-MS. Use of the Biochip to purify transthyretin from human serum allowed a distinct resolution of four disulfide conjugates and one truncated form isoforms with good mass resolution and sensitivity.

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific 'paints' from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10-12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.

A chemical affinity system exhibiting antibody-like properties is described. The system exploits bioconjugates with appended phenylboronic acid (PBA) moieties and a support-bound phenylboronic acid complexing reagent derived from salicylhydroxamic acid (SHA) for protein immobilization on a chromatographic support. The structure of the PBA.SHA complex was characterized by 11B NMR and mass spectrometry and compared with complexes derived from model compounds. Protein modification reagents were synthesized from 3-aminophenylboronic acid and utilized to prepare bioconjugates from alkaline phosphatase (AP) and horseradish peroxidase (HRP). AP obtained from one source afforded PBA bioconjugates exhibiting significant loss of enzymatic activity, whereas AP obtained from a second source afforded PBA bioconjugates exhibiting only a modest loss of enzymatic activity. Conversely, HRP afforded PBA bioconjugates exhibiting no loss of enzymatic activity. SHA-modified Sepharose was prepared by reaction of methyl 4-[(6-aminohexanoylamino)methyl]salicylate with CNBr-activated Sepharose 4B, followed by treatment with aqueous alkaline hydroxylamine. PBA-AP and PBA-HRP conjugates were efficiently immobilized on SHA-Sepharose at pH 8.3. PBA-AP conjugates were retained after washing with acidic buffers at pH 6.7, 4.2, and 2.5, whereas PBA-HRP conjugates were retained after washing with buffer at pH 6.7, but were eluted to some extent at and below pH 4.2. The results are interpreted in terms of multivalent interactions involving boronic acid complex formation between the enzyme bioconjugates and immobilized complexing reagent.

Over the course of the last decade, a number of investigators have come to appreciate that the surface of a MALDI target, after suitable modification, can be used for selective enrichment of peptides and proteins. More recently, surface-modified nanoparticles (NPs) that readily co-crystallize in MALDI matrix, are not ionized by laser desorption/ionization, and do not interfere with MS have attracted interest as alternatives to surface-modified targets for selective enrichment of peptides and proteins. Surface-modified targets and NPs facilitate parallel processing of samples, and when used in conjunction with MALDI mass spectrometers with kHz lasers enable development of high-throughput proteomics platforms. Targets and NPs for reversed phase and ion exchange retention, selective enrichment of glycopeptides, selective enrichment of phosphopeptides, and immunoaffinity MS are described in conjunction with details regarding their preparation and utility. Commercial availability of the reagents and substrates required to prepare surface-modified targets and NPs is also discussed.

Chemical protein sequencing remains the cornerstone of protein structural analysis. Recent developments allow an increased rate of sampling and a reduction in background noise. Mass spectrometric detection of chemical sequencing products will, in all likelihood, initiate an era of sequencing of sub-picomole quantities of protein. Significant progress toward routine, automated carboxy-terminal sequencing has been reported recently.

Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-11 residues as well as regional surface amino acids (Glu-23, Arg-32, and Arg-44) are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.

Publisher Summary The chapter describes the construction of a unique solid-phase protein sequencer having the capability to deliver reagents at precise flow rates with a single syringe-pump employed as a pneumatic displacement device. The instrument utilizes solenoid valves. The chapter illustrates the principles of the single syringe-pump concept. Initially, the syringe is filled with argon gas, which is metered by a pressure regulator through a port on a Rusnak valve. Any one of the six reagents that access the Hexagonal valve may be metered into a 500 μl volumetric loop situated between the Rusnak and Hexagonal valves. The volumetric loop may be partially filled or completely filled to waste via the second port on a Rusnak valve. The reagent is delivered to the reaction cartridge in a flow-rate dependent manner by displacing the contents of the volumetric loop with argon gas delivered from the syringe. The software driver allows flow rates to be corrected for solvent compressibility. Although compressibility correction factors can be calculated for pure solvents, they must be determined experimentally for solvent mixtures.

The thiobenzoylation method of protein sequencing was first proposed in 1967 by Barrett as an alternative to the Edman degradation (Edman, 1950). This method attracted limited interest but facilitated the subsequent development of the closely-related thioacetylation method (Doolittle et al., 1977), wherein the 2-methyl-5(4H)-thiazolone cleavage product is identified by back-hydrolysis to the parent amino acid. The 2-methyl-5(4H)-thiazolone is a volatile analyte which was shown by Previero in 1977 to be potentially suitable for analysis by gas chromatography, either directly or preferably after reaction with an acetylating reagent which affords the corresponding 5-acetoxy-2-methylthiazole. This proposed use of gas chromatography preceded the development of modern capillary gas chromatography columns by several years, however, and consequently column packings which permitted recovery of basic amino acid derivatives were unavailable. We recently reexamined this approach utilizing a capillary gas chromatography/mass spectrometry system and found the method to exhibit considerable potential (Stolowitz et al., 1992). Unfortunately, our efforts to further develop the thioacetylation method have been somewhat hindered by the limited reactivity of thioacetylating reagents and difficulties encountered in the synthesis of a few of the amino acid derivatives for use as analytical standards.

We describe a series of experiments that aimed to establish whether nuclease activity is actually associated with diphtheria toxin (DTx) and its A subunit (DTA), as we originally reported (M. P. Chang, R. L. Baldwin, C. Bruce, and B. J. Wisnieski, Science 246:1165-1168, 1989). Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration. In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity. These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx.

A reinvestigation of the thioacetylation method of protein sequencing ( G. A. Mross and R. F. Doolittle (1971 ) Fed. Proc. 30, 1241. G. A. Mross and R. F. Doolittle (1977) in Advanced Methods in Protein Sequence Determination (Needleman, S. B., Ed.), pp. 1–20, Springer, Berlin) has revealed that 2-methyl-5(4 H )-thiazolones, prepared by trifluoroacetic acid-catalyzed cleavage of the N-terminal amino acid from a N -thioacetylated polypeptide, were found to react instantaneously with one equivalent of carboxylic acid chloride, sulfonic acid chloride, or chloroformate to yield stable derivatives suitable for identification by high-performance liquid chromatography. NMR studies confirmed the products of the derivatization to be the corresponding 5- O -substituted-2-methylthiazoles. 2-Methyl-5(4 H )-thiazolones were derivatized by reaction with 3,5-dinitrobenzoyl chloride, 4-nitrophenylchloroformate, 4-nitrobenzenesulfonyl chloride, or 4- N -dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl chloride) in dichloromethane in the presence of triethylamine. Analytical standards were prepared by 1,3-dicyclohexylcarbodiimide-catalyzed cyclization of N -thioacetyl amino acids to 2-methyl-5(4 H )-thiazolones followed by derivatization with 4-nitrobenzenesulfonyl chloride. Stable crystalline 2-methyl-5- O -(4′-nitrobenzenesulfonyl)thiazole standards were obtained for 15 amino acids. Cysteine, serine, and threonine proved recalcitrant toward derivatization with 4-nitrobenzenesulfonyl chloride due to the dehydration of their respective thiazolones. Alkylated cysteine derivatives including ( S -β-(4-pyridylethyl)cysteine and S -ethylcysteine were derivatized without difficulty. Cyclization of N -thioacetylproline afforded a mesoionic compound which resisted derivatization, but could be detected directly. A preliminary high-performance liquid chromatographic separation was developed and the feasibility of this approach to protein sequencing demonstrated by solid-phase degradation of the oxidized insulin B chain. An average repetitive efficiency of 95% was obtained for 20 cycles of manual degradation. As compared to the products of the Edman degradation, 2-methyl-5- O -(4′-nitrobenzenesulfonyl)thiazole derivatives were obtained free of interferences arising during the coupling reaction.