We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1⌬ and xrn1⌬ mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5 to 3 decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression. Molecular & Cellular Proteomics 3:1154 -1169, 2004.An initial step in the systematic investigation of cellular processes is the identification and measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, however, a number of limitations imposed by mass-difference labeling. The mass-difference concept for many practical purposes is limited to a binary (2-plex) set of reagents, and this makes comparison of multiple states (e.g. several experimental controls or time-course studies) difficult to undertake. Multiple 2-plex datasets can be combined after separate analyses, but there is a high likelihood that different sets of peptides and proteins will be identified between each experiment. In addition, the use of massdifference labels increases MS complexity, and this problem increases with numbers of a multiplexed set. Finally, the cysteine-selective affinity strategy for reduction of sample complexity (ICAT) is not amenable to identification of post-translationally modified peptides, as the majority of posttranslational modification (PTM) 1 -containing peptides are discarded at the affinity step.We have developed a multiplexed set of reagents for quantitative protein analysis that place isobaric mass label...

The transcriptional network of the SRY (sex determining region Y)-box 17 (SOX17) and the prognostic impact of SOX17 protein expression in human cancers remain largely unclear. In this study, we evaluated the prognostic effect of low SOX17 protein expression and its dysregulation of transcriptional network in esophageal squamous cell carcinoma (ESCC). Low SOX17 protein expression was found in 47.4% (73 of 154) of ESCC patients with predicted poor prognosis. Re-expression of SOX17 in ESCC cells caused reduced foci formation, cell motility, decreased ESCC xenograft growth and metastasis in animals. Knockdown of SOX17 increased foci formation in ESCC and normal esophageal cells. Notably, 489 significantly differential genes involved in cell growth and motility controls were identified by expression array upon SOX17 overexpression and 47 genes contained putative SRY element in their promoters. Using quantitative chromatin immunoprecipitation-PCR and promoter activity assays, we confirmed that MACC1, MALAT1, NBN, NFAT5, CSNK1A1, FN1 and SERBP1 genes were suppressed by SOX17 via the SRY binding-mediated transcriptional regulation. Overexpression of FN1 and MACC1 abolished SOX17-mediated migration and invasion suppression. The inverse correlation between SOX17 and FN1 protein expression in ESCC clinical samples further strengthened our conclusion that FN1 is a transcriptional repression target gene of SOX17. This study provides compelling clinical evidence that low SOX17 protein expression is a prognostic biomarker and novel cell and animal data of SOX17-mediated suppression of ESCC metastasis. We establish the first transcriptional network and identify new suppressive downstream genes of SOX17 which can be potential therapeutic targets for ESCC.

Intervertebral disc degeneration (IDD) has been considered as the primary pathological mechanism that underlies low back pain. Understanding the molecular mechanisms underlying human IDD is imperative for making strategies to treat IDD-related diseases. Herein, we report the molecular programs, lineage progression patterns, and paths of cellular communications during the progression of IDD using single-cell RNA sequencing (scRNA-seq) on nucleus pulposus (NP) cells from patients with different grades of IDD undergoing discectomy. New subtypes of cells and cell-type-specific gene signatures of the metabolic homeostatic NP cells (Met NPC), adhesive NP cells (Adh NPC), inflammatory response NP cells (IR NPC), endoplasmic reticulum stress NP cells (ERS NPC), fibrocartilaginous NP cells (Fc NPC), and CD70 and CD82+ progenitor NP cells (Pro NPC) were identified. In the late stage of IDD, the IR NPC and Fc NPC account for a large proportion of NPC. Importantly, immune cells including macrophages, T cells, myeloid progenitors, and neutrophils were also identified, and further analysis showed that significant intercellular interaction between macrophages and Pro NPC occurred via MIF (macrophage migration inhibitory factor) and NF-kB signaling pathways during the progression of IDD. In addition, dynamic polarization of macrophage M1 and M2 cell subtypes was found in the progression of IDD, and gene set functional enrichment analysis suggested a significant role of the macrophage polarization in regulating cell metabolism, especially the Pro NPC. Finally, we found that the NP cells in the late degenerative stage were mainly composed of the cell types related to inflammatory and endoplasmic reticulum (ER) response, and fibrocartilaginous activity. Our results provided new insights into the identification of NP cell populations at single-cell resolution and at the relatively whole-transcriptome scale, accompanied by cellular communications between immune cells and NP cells, and discriminative markers in relation to specific cell subsets. These new findings present clues for effective and functional manipulation of human IDD-related bioremediation and healthcare.

Manuscript Type: EmpiricalResearch Question/Issue: This study distinguishes between the effects of financial constraint determinants on cross-border mergers and acquisitions (M&As) and domestic M&As for all takeover bids announced in nine East Asian economies from 1998 to 2005. Research Findings/Insights: The results of logistic regressions verify that the extent of stock market and governance developments improves corporate financing conditions and subsequently encourages cross-border M&As in East Asia. The results also indicate that, except for ownership control variables, the firm-specific factors of financing constraints reduce the occurrence of cross-border M&As relative to domestic M&As. Although family-and state-controlled firms have better access to external financing, they are reluctant to risk diluting their management control and thus prefer domestic M&As to cross-border deals. Theoretical/Academic Implications: This study enhances the empirical studies of the relation between financing constraints and corporate investments based on the market imperfection hypothesis of corporate finance theories. In addition, this study also addresses the interaction between the market imperfection hypothesis and agency theory in explaining the effects of special ownership control on cross-border M&As relative to domestic deals. Furthermore, by examining the research questions across nine East Asian economies, this study provides an understanding of how such a relation applies to firms in countries where information asymmetry is high. Practitioner/Policy Implications: The findings indicate the importance of corporate governance and verify the effects of unique organizational structures on major corporate decisions. Specifically, family-controlled firms are often free of the financing constraints inherent in investment decisions. Thus, it is necessary to consider such organizational uniqueness when explaining the financing behavior of cross-border M&As conducted by Asian firms.