Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz.

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes. Here, we report the identification of Cup as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end-directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (eIF4E) is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. We propose that Cup coordinates localization with translation.

Signal peptide prediction based on analysis of experimentally verified cleavage sites.

A number of computational tools are available for detecting signal peptides, but their abilities to locate the signal peptide cleavage sites vary significantly and are often less than satisfactory. We characterized a set of 270 secreted recombinant human proteins by automated Edman analysis and used the verified cleavage sites to evaluate the success rate of a number of computational prediction programs. An examination of the frequency of amino acid in the N-terminal region of the data set showed a preference of proline and glutamine but a bias against tyrosine. The data set was compared to the SWISS-PROT database and revealed a high percentage of discrepancies with cleavage site annotations that were computationally generated. The best program for predicting signal sequences was found to be SignalP 2.0-NN with an accuracy of 78.1% for cleavage site recognition. The new data set can be utilized for refining prediction algorithms, and we have built an improved version of profile hidden Markov model for signal peptides based on the new data.

Protein identification: the origins of peptide mass fingerprinting.

Peptide mass fingerprinting (PMF) grew from a need for a faster, more efficient method to identify frequently observed proteins in electrophoresis gels. We describe the genesis of the idea in 1989, and show the first demonstration with fast atom bombardment mass spectrometry. Despite its promise, the method was seldom used until 1992, with the coming of significantly more sensitive commercial instrumentation based on MALDI-TOF-MS. We recount the evolution of the method and its dependence on a number of technical breakthroughs, both in mass spectrometry and in other areas. We show how it laid the foundation for high-throughput, high-sensitivity methods of protein analysis, now known as proteomics. We conclude with recommendations for further improvements, and speculation of the role of PMF in the future.

Asparagine deamidation in recombinant human lymphotoxin: hindrance by three-dimensional structures.

The chemical stability of recombinant human lymphotoxin (rhLT) was evaluated at pH 7, 9, and 11 and 40 degrees C using quantitative tryptic map and urea-IEF methods. Degradation products were characterized by mass spectrometry. The stability of denatured rhLT protein was also evaluated to elucidate the effects of three-dimensional structures on Asn deamidation in rhLT. Two sites that underwent Asn deamidation were identified in rhLT, Asn(19) and Asn(40)-Asn(41). At pH 11 and 40 degrees C, deamidation at Asn(19) and Asn(40)-Asn(41) had half-lives of 14 +/- 4 and 80 +/- 24 days, respectively. Upon denaturation, 31- and ninefold acceleration in the degradation rates was observed at the Asn(19) and Asn(40)-Asn(41) sites, respectively. The rate of Asn(19) degradation in denatured rhLT was comparable to that of the model peptide possessing the same primary sequence as the Asn(19)-containing region in rhLT. Analysis of the rhLT crystal structure revealed that both Asn deamidation sites were located in beta-turn structures with extensive hydrogen-bonding networks created with nearby residues in the tertiary structures. The results suggested that these tertiary and secondary structures, if held true in solution, were probably responsible for the stabilization of Asn in the native rhLT protein by reducing flexibility, thus preventing adoption of the favorable conformation required for cyclic-imide formation.

De novo proteomic sequencing of a monoclonal antibody raised against OX40 ligand.

De novo sequencing of a full-length monoclonal antibody raised against OX40 ligand is described. Using a combination of overlapping complementary proteolytic and chemical digestions, with analysis by mass spectrometry and Edman degradation, both the heavy and light chains were fully sequenced. Particular attention was paid to those modifications that could be susceptible to degradation in the complementarity determining region and Fc region. An overview of the protocol is described, and suggestions for improvements to aid in such sequencing projects in the future are discussed.

Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins.

A method for the rapid limited enzymatic cleavage of PVDF membrane-immobilized proteins is described. This method allows the fast characterization of PVDF blotted proteins by peptide mass fingerprinting (Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., Wantanabe, C., Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015), LC-MS/MS, or N-terminal sequencing and has been demonstrated on a range of proteins using a full complement of proteolytic enzymes. This technique allows the generation of proteolytic fragments between 5 and 60 min (depending on the enzyme employed), which is significantly faster than previously reported on-membrane digestion methods. To date, this on-membrane rapid digestion protocol has aided in the identification and confirmation of mutation sites in over 200 recombinant proteins.

Reversed-phase isolation of peptides.

Reversed-phase high-performance liquid chromatography (HPLC) is a fundamental tool for the isolation and analysis of peptides. Peptides are separated on a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent concentration. This unit presents protocol for separation of 5- to 500-pmol of peptides on a narrow-bore (2-mm-i.d.) or microbore (1-mm-i.d.) column. Smaller quantities can be separated capillary HPLC columns, as described. Capillary HPLC columns, however, require a gradient flow rate of 3 to 5 ml/min, which most current HPLC pumps cannot attain without modifications. A procedure is therefore provided for constructing a capillary HPLC system using readily available components. HPLC peaks that appear to be symmetrical may actually contain coeluting peptides. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and capillary electrophoresis are described for analysis of a small portion of an HPLC fraction to determine the number of components present in a small sample. These methods can be utilized to screen fractions prior to automated sequencing.

High-throughput protein sequencing.

We have designed and implemented an autosampler that provides additional sample capacity on a commercial protein sequencer. The autosampler attaches to a standard ABI Procise sequencer, enabling a single-sample cartridge to hold up to six separate samples. The autosampler is used in combination with faster Edman cycles and a rapid 12-min PTH separation to significantly increase the speed of automated protein sequencing. We also describe Edman chemistry modifications that improve coupling efficiency and prevent internal cleavage that can occur when samples are sequenced in the standard glass cartridges.