The identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%-100%). In addition, we developed a respiration-based fully automated non-invasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research.

Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotopelabeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation.absolute quantification | mass spectrometry | cell-free protein synthesis system | mammalian circadian clock protein | targeted proteomics Q uantitative information on protein expression levels is important to define the dynamic state of cells. Accurate measurements of absolute protein abundance in cells can be performed by emerging quantitative proteomics approaches such as selected reaction monitoring (SRM) or high-resolution mass spectrometry (MS) in combination with isotope dilution strategies (1). The methods require a known concentration of internal standards, typically prepared as the tryptic digests of target proteins, which are labeled with isotopically heavy atoms. The standard peptides are combined with samples containing the same peptides, and the mixtures are analyzed by MS. Quantities of peptides, which represent the target proteins, can be calculated by comparing ion intensities for isotopically light and heavy peptides.The preparation of isotope-labeled peptides plays a major role in these approaches. The most common way to prepare such peptides is by absolute quantification, which uses chemical synthesis of peptides containing isotopically labeled amino acids (1, 2). It provides not only normal proteotypic peptides, but also modified peptides that mimic posttranslational modification. However, the method has several limitations originating from the use of the chemical synthesis. It requires individual peptide synt...

To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1:Cry2 background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.

Protein phosphorylation is a key mechanism of cellular signaling pathways and aberrant phosphorylation has been implicated in a number of human diseases. Thus, approaches in phosphoproteomics can contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets. Moreover, careful validation of large-scale phosphoproteome analysis, which is lacking in the current protein-based biomarker discovery, significantly increases the value of identified biomarkers. Here, we performed large-scale differential phosphoproteome analysis using IMAC coupled with the isobaric tag for relative quantification (iTRAQ) technique and subsequent validation by selected/multiple reaction monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk recurrence groups. We identified 8309 phosphorylation sites on 3401 proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were able to be quantified and 133 phosphopeptides (117 phosphoproteins) were differentially expressed between the two groups. Among them, 19 phosphopeptides were selected for further verification and 15 were successfully quantified by SRM using stable isotope peptides as a reference. The ratio of phosphopeptides between high- and low-risk groups quantified by SRM was well correlated with iTRAQ-based quantification with a few exceptions. These results suggest that large-scale phosphoproteome quantification coupled with SRM-based validation is a powerful tool for biomarker discovery using clinical samples.

Recent advances in quantitative proteomic technology have enabled the large-scale validation of biomarkers. We here performed a quantitative proteomic analysis of membrane fractions from colorectal cancer tissue to discover biomarker candidates, and then extensively validated the candidate proteins identified. A total of 5566 proteins were identified in six tissue samples, each of which was obtained from polyps and cancer with and without metastasis. GO cellular component analysis predicted that 3087 of these proteins were membrane proteins, whereas TMHMM algorithm predicted that 1567 proteins had a transmembrane domain. Differences were observed in the expression of 159 membrane proteins and 55 extracellular proteins between polyps and cancer without metastasis, while the expression of 32 membrane proteins and 17 extracellular proteins differed between cancer with and without metastasis. A total of 105 of these biomarker candidates were quantitated using selected (or multiple) reaction monitoring (SRM/MRM) with stable synthetic isotope-labeled peptides as an internal control. The results obtained revealed differences in the expression of 69 of these proteins, and this was subsequently verified in an independent set of patient samples (polyps (n ‫؍‬ 10), cancer without metastasis (n ‫؍‬ 10), cancer with metastasis (n ‫؍‬ 10)). Significant differences were observed in the expression of 44 of these proteins, including ITGA5, GPRC5A, PDGFRB, and TFRC, which have already been shown to be overexpressed in colorectal cancer, as well as proteins with unknown function, such as C8orf55. The expression of C8orf55 was also shown to be high not only in colorectal cancer, but also in several cancer tissues using a multicancer tissue microarray, which included 1150 cores from 14 cancer tissues. This is the largest verification study of biomarker candidate membrane proteins to date; our methods for biomarker discovery and subsequent validation using SRM/MRM will contribute to the identification of useful biomarker candidates for various cancers. Recent advances in proteomic technology have contributed to the identification of biomarkers for various diseases. Improvements in LC-MS technology have led to an increase in the number of proteins that have been identified. In addition, a stable isotopic labeling method using isobaric tag for relative and absolute quantitation (iTRAQ) 1 and stable isotope labeling by amino acids in cell culture has enabled the quantitative analysis of multiple samples (1, 2). Therefore, a large From the ‡Laboratory