Analyzing LC-MS/MS data by spectral count and ion abundance: two case studies.

In comparative proteomics studies, LC-MS/MS data is generally quantified using one or both of two measures: the spectral count, derived from the identification of MS/MS spectra, or some measure of ion abundance derived from the LC-MS data. Here we contrast the performance of these measures and show that ion abundance is the more sensitive. We also examine how the conclusions of a comparative analysis are influenced by the manner in which the LC-MS/MS data is 'rolled up' to the protein level, and show that divergent conclusions obtained using different rollups can be informative. Our analysis is based on two publicly available reference data sets, BIATECH-54 and CPTAC, which were developed for the purpose of assessing methods used in label-free differential proteomic studies. We find that the use of the ion abundance measure reveals properties of both data sets not readily apparent using the spectral count.

Exposed hydrophobicity is a key determinant of nuclear quality control degradation.

Protein quality control (PQC) degradation protects the cell by preventing the toxic accumulation of misfolded proteins. In eukaryotes, PQC degradation is primarily achieved by ubiquitin ligases that attach ubiquitin to misfolded proteins for proteasome degradation. To function effectively, PQC ubiquitin ligases must distinguish misfolded proteins from their normal counterparts by recognizing an attribute of structural abnormality commonly shared among misfolded proteins. However, the nature of the structurally abnormal feature recognized by most PQC ubiquitin ligases is unknown. Here we demonstrate that the yeast nuclear PQC ubiquitin ligase San1 recognizes exposed hydrophobicity in its substrates. San1 recognition is triggered by exposure of as few as five contiguous hydrophobic residues, which defines the minimum window of hydrophobicity required for San1 targeting. We also find that the exposed hydrophobicity recognized by San1 can cause aggregation and cellular toxicity, underscoring the fundamental protective role for San1-mediated PQC degradation of misfolded nuclear proteins.

Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates.

Protein quality control (PQC) degradation systems protect the cell from the toxic accumulation of misfolded proteins. Because any protein can become misfolded, these systems must be able to distinguish abnormal proteins from normal ones, yet be capable of recognizing the wide variety of distinctly shaped misfolded proteins they are likely to encounter. How individual PQC degradation systems accomplish this remains an open question. Here we show that the yeast nuclear PQC ubiquitin ligase San1 directly recognizes its misfolded substrates via intrinsically disordered N- and C-terminal domains. These disordered domains are punctuated with small segments of order and high sequence conservation that serve as substrate-recognition sites San1 uses to target its different substrates. We propose that these substrate-recognition sites, interspersed among flexible, disordered regions, provide San1 an inherent plasticity which allows it to bind its many, differently shaped misfolded substrates.

Maximal power tests for detecting defects in meiotic recombination.

We have determined the marker separations (genetic distances) that maximize the probability, or power, of detecting meiotic recombination deficiency when only a limited number of meiotic progeny can be assayed. We find that the optimal marker separation is as large as 30-100 cM in many cases. Provided the appropriate marker separation is used, small reductions in recombination potential (as little as 50%) can be detected by assaying a single interval in as few as 100 progeny. If recombination is uniformly altered across the genomic region of interest, the same sensitivity can be obtained by assaying multiple independent intervals in correspondingly fewer progeny. A reduction or abolition of crossover interference, with or without a reduction of recombination proficiency, can be detected with similar sensitivity. We present a set of graphs that display the optimal marker separation and the number of meiotic progeny that must be assayed to detect a given recombination deficiency in the presence of various levels of crossover interference. These results will aid the optimal design of experiments to detect meiotic recombination deficiency in any organism.