Stephen A. Martin

Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Δ and xrn1Δ mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5′ to 3′ decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.

On the initial velocity of ions generated by matrix-assisted laser desorption ionization and its effect on the calibration of delayed extraction time-of-flight mass spectra

A novel method was developed to measure the initial velocity of ions generated by matrix-assisted laser desorption ionization (MALDI). It is shown both experimentally and theoretically that with a delayed extraction (DE) technique, the flight time of an ion changes linearly with extraction delay. The initial velocity of the ion, a consequence of the desorption process, can be determined from the slope of this linear curve. Systematic study of the initial velocity was undertaken regarding its dependence on the matrix substance, molecular weight of the analyte, ion polarity, and wavelength of irradiation. It was found that the most important factor was the matrix material. Sinapinic acid and α-cyano-4-hydroxycinnamic acid matrices ejected slower peptide and protein ions than 2,5-dihydroxybenzoic acid or 3-hydroxypicolinic acid: ∼ 300 versus ∼ 550 m/s. Matrix ions themselves exhibited a similar order of initial velocities, but these were 15–40% higher than those of insulin ions. The molecular weight of protein samples (between 5 and 25 ku) was found to have little effect on the initial velocity, but for peptides below 5 ku a gradual transition was noted toward the velocity of the matrix ions. Also decreasing velocity with increasing molecular mass was observed for DNA samples in the 4–14-ku range. In the negative ion mode slightly lower velocities were observed than in the positive ion mode. No difference was found between 337- and 266-nm irradiation. Values of the initial velocities were used to correct systematic errors in the internal calibration observed in mass spectra with delayed extraction. These velocity corrections decrease mass errors substantially in the linear mode, in particular for multicomponent mixtures.

Accurate Mass Measurements Using MALDI-TOF with Delayed Extraction

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100–500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.

The utility of nonspecific proteases in the characterization of glycoproteins by high-resolution time-of-flight mass spectrometry

Abstract Degradation of glycoproteins with the nonspecific protease pronase was examined by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS). High mass resolution (in excess of 10 000 at FWHM) and mass accuracy on monoisotopic species (better than 10 ppm) obtained with the combination of delayed extraction and the reflector mode of analysis enabled the successful interpretation of very complex mixtures resulting from extensive hydrolysis. After 48 h of degradation of glycoproteins, the glycopeptides were well separated from small peptides based on their molecular weight. Accurate monoisotopic masses of the glycopeptides permitted the determination of the glycan composition and the (nonspecific) peptide segment the glycans were attached to. Ribonuclease B and ovalbumin were used to demonstrate feasibility of the method. Unless the peptide sequence of the glycopeptide contains a basic residue, the negative-ion mode is preferred over the positive-ion mode. The absence of alkali adducts reduce the complexity of the mass spectra and the relative sensitivities of the glycopeptides were found better in the negative-ion mode. A highly complex sample, α-acid glycoprotein was also analyzed. Multiply sialylated glycopeptides had to be run in the linear mode where the metastable loss of sialic acid residues did not interfere with the detection of other components. From the linear mode mass spectrum, five glycosylation sites were identified along with the more abundant glycoforms. Comparison with the literature indicated that several minor components were undetected. The presented approach also permitted the determination of the sialylation pattern of the complex type glycans.

Construction of a large extracellular protein interaction network and its resolution by spatiotemporal expression profiling.

Extracellular interactions involving both secreted and membrane-tethered receptor proteins are essential to initiate signaling pathways that orchestrate cellular behaviors within biological systems. Because of the biochemical properties of these proteins and their interactions, identifying novel extracellular interactions remains experimentally challenging. To address this, we have recently developed an assay, AVEXIS (avidity-based extracellular interaction screen) to detect low affinity extracellular interactions on a large scale and have begun to construct interaction networks between zebrafish receptors belonging to the immunoglobulin and leucine-rich repeat protein families to identify novel signaling pathways important for early development. Here, we expanded our zebrafish protein library to include other domain families and many more secreted proteins and performed our largest screen to date totaling 16,544 potential unique interactions. We report 111 interactions of which 96 are novel and include the first documented extracellular ligands for 15 proteins. By including 77 interactions from previous screens, we assembled an expanded network of 188 extracellular interactions between 92 proteins and used it to show that secreted proteins have twice as many interaction partners as membrane-tethered receptors and that the connectivity of the extracellular network behaves as a power law. To try to understand the functional role of these interactions, we determined new expression patterns for 164 genes within our clone library by using whole embryo in situ hybridization at five key stages of zebrafish embryonic development. These expression data were integrated with the binding network to reveal where each interaction was likely to function within the embryo and were used to resolve the static interaction network into dynamic tissue- and stage-specific subnetworks within the developing zebrafish embryo. All these data were organized into a freely accessible on-line database called ARNIE (AVEXIS Receptor Network with Integrated Expression; www.sanger.ac.uk/arnie) and provide a valuable resource of new extracellular signaling interactions for developmental biology.

Depth of Proteome Issues A Yeast Isotope-Coded Affinity Tag Reagent Study

As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system (Gygi et al. (1999) Nat. Biotechnol. 17, 994–999) was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to “single hit” proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.

Multidimensional chromatography coupled with mass spectrometry for target-based screening

SummaryThe synthesis of structural analogs and the process of drug discovery have evolved dramatically through recent advances in solid-phase synthesis reagents and automated screening systems. As molecular diversity strategies emerge, the need for automated target-based selection of lead candidates becomes equally important. Multidimensional automated chromatographic techniques coupled to electrospray ionization mass spectrometry facilitate the selection process and provide maximum characterization information in a single screening run. The capture of tightly bound affinity leads by target biomolecules, followed by subsequent release and high-resolution separation with sensitive detection, significantly reduces the time required to identify and characterize lead compounds. This automated multidimensional chromatographic approach coupled with mass spectrometry, Selectronics™, was used with several organic and natural libraries to demonstrate an automated target-based screening technique to select for high-affinity binders as potential lead compounds.

Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI MS/MS.

A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.

A New Delayed Extraction MALDI-TOF MS-MS for Characterization of Protein Digests

Two-dimensional gel electrophoresis is commonly used to separate and visualize proteins present in complex mixtures such as cell lysates [1]. Digestion of selected spots by one or more endopeptidases followed by generation of peptide mass maps using MALDI-TOF MS is now widely accepted as the first step toward identifying and characterizing the proteins [2]. Following the development of delayed extraction techniques for MALDI [3–5], and improved sample preparation and clean-up methodology [6], this strategy is often successful at identifying proteins represented in a database [7] even when the proteins are present at low levels. In favorable cases protein identification is successful at the sub-femtomole level. When this approach fails, it is generally necessary to generate sequence data using either Edman degradation or MS-MS techniques. The MS-MS technique known as post-source decay (PSD) with MALDI-TOF [8] sometimes provides sufficient sequence information, but often the sensitivity and mass accuracy are inadequate and interpretation is difficult. Recent developments in electrospray ionization, such as nanospray [9] have dramatically improved the sensitivity of triple quadrupole and ion trap MS-MS, but these techniques are rather slow and tedious, and rapid, automated interpretation of data to provide reliable sequences is not yet routine. New instruments employing TOF analyzers in place of the third quadrupole in triple quadrupole MS-MS systems have very recently been described which provide improved resolution and mass accuracy in fragment ion measurements [10, 11].

High-throughput identification of transient extracellular protein interactions.

Protein interactions are highly diverse in their biochemical nature, varying in affinity and are often dependent on the surrounding biochemical environment. Given this heterogeneity, it seems unlikely that any one method, and particularly those capable of screening for many protein interactions in parallel, will be able to detect all functionally relevant interactions that occur within a living cell. One major class of interactions that are not detected by current popular high-throughput methods are those that occur in the extracellular environment, especially those made by membrane-embedded receptor proteins. In the present article, we discuss some of our recent research in the development of a scalable assay to identify this class of protein interaction and some of the findings from its application in the construction of extracellular protein interaction networks.