Protein significance analysis in selected reaction monitoring (SRM) measurements

Selected reaction monitoring (SRM) is a targeted mass spectrometry technique that provides sensitive and accurate protein detection and quantification in complex biological mixtures. Statistical and computational tools are essential for the design and analysis of SRM experiments, particularly in studies with large sample throughput. Currently, most such tools focus on the selection of optimized transitions and on processing signals from SRM assays. Little attention is devoted to protein significance analysis, which combines the quantitative measurements for a protein across isotopic labels, peptides, charge states, transitions, samples, and conditions, and detects proteins that change in abundance between conditions while controlling the false discovery rate. We propose a statistical modeling framework for protein significance analysis. It is based on linear mixed-effects models and is applicable to most experimental designs for both isotope label-based and label-free SRM workflows. We illustrate the utility of the framework in two studies: one with a group comparison experimental design and the other with a time course experimental design. We further verify the accuracy of the framework in two controlled data sets, one from the NCI-CPTAC reproducibility investigation and the other from an in-house spike-in study. The proposed framework is sensitive and specific, produces accurate results in broad experimental circumstances, and helps to optimally design future SRM experiments. The statistical framework is implemented in an open-source R-based software package SRMstats, and can be used by researchers with a limited statistics background as a stand-alone tool or in integration with the existing computational pipelines.     

Contrasted patterns of mitochondrial and nuclear structure among nursery colonies of the bat Myotis myotis

Thirteen nursery colonies of Myotis myotis were sampled in central Europe to investigate the dispersal behaviour of this bat species. Mitochondrial DNA sequences of 260 bats reveal the occurrence of three evolutionary lineages that have probably originated in distinct glacial refugia and meet in a contact zone near the Alps. Moreover, the strong haplotypic segregation (Φ_ST=0.540) suggests that breeding females are philopatric. Contrastingly, the low population structure at 15 microsatellite loci (F_ST=0.022), suggests the homogenizing effect of nuclear gene flow. The different perspectives given by these two markers are consistent with strong male‐biased dispersal. As a result of female philopatry, the local haplotypic variability seems to be largely influenced by historical processes of colonization. Conversely, the homogeneity of nuclear variability within roosts that are located north of the Alps seems to mainly reflect contemporary gene flow. Finally, despite the fact that females are faithful to their natal colony, movements of both males and females occur outside the breeding period. Mitochondrial survey of individuals sampled exclusively in nurseries may thus poorly reflect the metapopulation dynamics of this species.     

Systematic characterization of pan‐cancer mutation clusters

Cancer genome sequencing has shown that driver genes can often be distinguished not only by the elevated mutation frequency but also by specific nucleotide positions that accumulate changes at a high rate. However, properties associated with a residue's potential to drive tumorigenesis when mutated have not yet been systematically investigated. Here, using a novel methodological approach, we identify and characterize a compendium of 180 hotspot residues within 160 human proteins which occur with a significant frequency and are likely to have functionally relevant impact. We find that such mutations (i) are more prominent in proteins that can exist in the on and off state, (ii) reflect the identity of a tumor of origin, and (iii) often localize within interfaces which mediate interactions with other proteins or ligands. Following, we further examine structural data for human protein complexes and identify a number of additional protein interfaces that accumulate cancer mutations at a high rate. Jointly, these analyses suggest that disruption and dysregulation of protein interactions can be instrumental in switching functions of cancer proteins and activating downstream changes.     

High-resolution, human–bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments. Received: 6 August 1998 / Accepted: 28 October 1998     

Construction and characterization of a porcine P1-derived artificial chromosome (PAC) library covering 3.2 genome equivalents and cytogenetical assignment of six type I and type II loci

A porcine P1-derived artificial chromosome (PAC) library of a male German Landrace pig was constructed in pCYPAC2. In total 90,240 clones were generated and individually transferred into microtiter plates. An average insert size of 119.1 kb was determined by analyzing 150 randomly selected PAC clones by pulsed field electrophoresis, yielding approximately 3.2 genome equivalents. The stability of nine clones was followed through 110 generations showing no reduction of the insert size. The probability of identifying a specific chromosomal region within the library was tested by screening for the presence of seven type I and five type II loci. The analysis showed that most loci (10/12) were present in the library at least twice. To determine the percentage of chimerism, six clones were analyzed by fluorescence in situ hybridization (FISH) on metaphase chromosomes. We assign one type I locus (Triadin) and three type II loci (SW855, S0300, SW1129). Received: 24 November 1998 / Accepted: 1 February 1999     

Proteome analysis of Halobacterium sp. NRC-1 facilitated by the biomodule analysis tool BMSorter

To better understand the extremely halophilic archaeon Halobacterium species NRC-1, we analyzed its soluble proteome by two-dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 888 unique proteins were identified with a ProteinProphet probability (P) between 0.9 and 1.0. To evaluate the biochemical activities of the organism, the proteomic data were subjected to a biological network analysis using our BMSorter software. This allowed us to examine the proteins expressed in different biomodules and study the interactions between pertinent biomodules. Interestingly an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle networks suggested that up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. In addition, glutamate and aspartate may be interconverted from other amino acids or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino acids that are required to build the highly acidic proteome of the organism. Thus this study demonstrated that proteome analysis can provide useful information and help systems analyses of organisms.