Robust and scalable single-molecule protein sequencing with fluorosequencing.

The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications.

GIANT EMBRYO encodes CYP78A13, required for proper size balance between embryo and endosperm in rice.

Among angiosperms there is a high degree of variation in embryo/endosperm size in mature seeds. However, little is known about the molecular mechanism underlying size control between these neighboring tissues. Here we report the rice GIANT EMBRYO (GE) gene that is essential for controlling the size balance. The function of GE in each tissue is distinct, controlling cell size in the embryo and cell death in the endosperm. GE, which encodes CYP78A13, is predominantly expressed in the interfacing tissues of the both embryo and endosperm. GE expression is under negative feedback regulation; endogenous GE expression is upregulated in ge mutants. In contrast to the loss-of-function mutant with large embryo and small endosperm, GE overexpression causes a small embryo and enlarged endosperm. A complementation analysis coupled with heterofertilization showed that complementation of ge mutation in either embryo or endosperm failed to restore the wild-type embryo/endosperm ratio. Thus, embryo and endosperm interact in determining embryo/endosperm size balance. Among genes associated with embryo/endosperm size, REDUCED EMBRYO genes, whose loss-of-function causes a phenotype opposite to ge, are revealed to regulate endosperm size upstream of GE. To fully understand the embryo-endosperm size control, the genetic network of the related genes should be elucidated.

SplitTester: software to identify domains responsible for functional divergence in protein family.

BACKGROUND: Many protein families have undergone functional divergence after gene duplications such that current subgroups of the family carry out overlapping but distinct biological roles. For the protein families with known functional subtypes (a functional split), we developed the software, SplitTester, to identify potential regions that are responsible for the observed distinct functional subtypes within the same protein family. RESULTS: Our software, SplitTester, takes a multiple protein sequences alignment as input, generated from protein members of two subgroups with known functional divergence. SplitTester was designed to construct the neighbor joining tree (a split cluster) from variable-sized sliding windows across the alignment in a process called split-clustering. SplitTester identifies the regions, whose split cluster is consistent with the functional split, but may be inconsistent with the phylogeny of the protein family. We hypothesize that at least some number of these identified regions, which are not following a random mutation process, are responsible for the observed functional split. To test our method, we used reverse transcriptase from a group of Pseudoviridae retrotransposons: to identify residues specific for diverged primer recognition. Candidate regions were then mapped onto the three dimensional structures of reverse transcriptase. The locations of these amino acids within the enzyme are consistent with their biological roles. CONCLUSION: SplitTester aims to identify specific domain sequences responsible for functional divergence of subgroups within a protein family. From the analysis of retroelements reverse transcriptase family, we successfully identified the regions splitting this family according to the primer specificity, implying their functions in the specific primer selection.

DIVERGE: phylogeny-based analysis for functional-structural divergence of a protein family.

SUMMARY: DetectIng Variability in Evolutionary Rates among GEnes (DIVERGE) is a software system to study functional divergence of a protein family by detecting site-specific change in evolutionary rate using a multiple alignment of amino acid sequences for a given phylogenetic tree. The program first conducts a statistical test for site-specific rate shifts along the tree, and predicting candidate amino acid residues responsible for functional divergence based on posterior analysis. These results can then be mapped on the 3D protein structure if available. AVAILABILITY: DIVERGE is available free of charge from http://xgu1.zool.iastate.edu/. Distribution packages for both Linux and Microsoft Windows operating systems are available, including manual and example files.