Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome

Kang C.; Huh S.; Nam D.; Kim H.; Hong J.; Hwang D.; Lee S.-W.; Anal. Chem., 94, 35, 12185–12195, 2022

Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. ...

Novel Diagnostic Biomarkers for High-Grade Serous Ovarian Cancer Uncovered by Data-Independent Acquisition Mass Spectrometry

Huh S.; Kang C.; Park J.E.; Nam D.; Kim S.I.; Seol A.; Choi K.; Hwang D.; Yu M.-H.; Chung H.H; Lee S.-W.; Kang U.-B.; J. Proteome Res., 21, 9, 2146–2159, 2022

High-grade serous ovarian cancer (HGSOC) represents the major histological type of ovarian cancer, and the lack of effective screening tools and early detection methods significantly contributes to the poor prognosis of HGSOC. Currently, there are no reliable diagnostic biomarkers for HGSOC. In this study, we performed liquid chromatography data-independent acquisition tandem mass spectrometry (MS) on depleted serum samples from 26 HGSOC cases and 24 healthy controls (HCs) to discover potential HGSOC diagnostic biomarkers. A total of 1,847 proteins were identified across all samples, among which 116 proteins showed differential expressions between HGSOC patients and HCs. ...

DNA repair and cholesterol-mediated drug efflux induce dose-dependent chemoresistance in nutrient-deprived neuroblastoma cells

Chae S.Y.; Nam D.; Hyeon D.Y.; Hong A.; Lee T.D.; Kim S.; Im D.; Hong J.; Kang C.; Lee J.W.; Hwang D.; Lee S.-W.; Kim H. I.; iScience 24, 4, 102325, 2021

Neuroblastoma is a solid, heterogeneous pediatric tumor. Chemotherapy is widely used to treat neuroblastoma. However, dose-dependent responses and chemoresistance mechanisms of neuroblastoma cells to anticancer drugs remain challenging. Here, we investigated the dose-dependent effects of topotecan on human neuroblastoma cells (SK-N-SH, SH-SY5Y, and SK-N-BE) under various nutrient supply conditions. Serum-starved human neuroblastoma cells showed reduced toxicity. Their survival rate increased upon treatment with a high concentration (1 μM) of topotecan. Quantitative profiling of global and phosphoproteome identified 12,959 proteins and 48,812 phosphosites, respectively, from SK-N-SH cells. ...

Blockers of Wnt3a, Wnt10a, or β-Catenin Prevent Chemotherapy-Induced Neuropathic Pain in Vivo Neurotherapeutics

Kim H.K.; Bae J.; Lee S.H.; Hwang S.-H.; Kim M.-S; Kim M.J.; Jun S.; Cervantes C.L.; Jung Y.-S.; Back S.; Lee H.; Lee S.-E.; Dougherty P.; Lee S.-W.; Park J.-I.; Abdi S.; Neurotherapeutics 18, 601–614, 2021

Although chemotherapy is a key cancer treatment, many chemotherapeutic drugs produce chronic neuropathic pain, called chemotherapy-induced neuropathic pain (CINP), which is a dose-limiting adverse effect. To date, there is no medicine that prevents CINP in cancer patients and survivors. We determined whether blockers of the canonical Wnt signaling pathway prevent CINP. Neuropathic pain was induced by intraperitoneal injection of paclitaxel (PAC) on four alternate days in male Sprague-Dawley rats or male Axin2-LacZ knock-in mice. XAV-939, LGK-974, and iCRT14, Wnt/β-catenin blockers, were administered intraperitoneally as a single or multiple doses before or after injury. ...

PASS-DIA: A data-independent acquisition approach for discovery studies

Mun D.-G.; Renuse S.; Saraswat M.; Madugund A.; Udainiya S.; Kim H.; Park S.-K.R.; Zhao H.; Nirujogi R.S.; Na C.H.; Kannan N.; Yates J.R.III.; Lee S.-W.; Pandey A.; Anal. Chem. 2020, 92, 21, 14466–14475

Data- independent acquisition (DIA) approach is being increasingly adopted as a promising strategy for identification and quantitation of proteomes. As most DIA datasets are acquired with wide isolation windows, highly complex MS/MS spectra are generated, which negatively impacts obtaining peptide information through classical protein database searches. Therefore, analysis of DIA data mainly relies on evidence of the existence of peptides from pre-built spectral libraries. Consequently, one major weakness of this method is that it does not account for peptides which are not included in spectral library, precluding the use of DIA for discovery studies. ...

Prognostic plasma protein panel for Aβ deposition in the brain in Alzheimer’s disease

Park, J.-C.; Han, S.-H.; Lee. H.; Jeong, H.; Byun, M. S.; Bae, J.; Kim, H.; Lee, D. Y.; Yi, D.; Shin, S. A.; Kim, Y. K.; Hwang, D.; Lee, S.-W.; Mook-Jung, I. Progress in Neurobiology 2019, 183, 1-13

Alzheimer’s disease (AD) is the most common age-associated dementia. Many studies have sought to predict cerebral amyloid deposition, the major pathological hallmark of AD, using body fluids such as blood or cerebral spinal fluid (CSF). The use of blood in diagnostic procedures is widespread in medicine; however, existing blood biomarkers for AD remain unreliable. We sought to discover blood biomarkers that discriminate Aβ deposition status in the brain. This study used 107 individuals who were cognitively normal (CN), ...

Accurate Precursor Mass Assignment Improves Peptide Identification in Data-Independent Acquisition Mass Spectrometry

Mun, D.-G.; Nam, D.; Kim, H.; Pandey, A.; Lee, S.-W. Anal. Chem. 2019, 91, 8453-8460

Proteomics research today no longer simply seeks exhaustive protein identification; increasingly, it is also desirable to obtain robust, large-scale quantitative information. To accomplish this, data-independent acquisition (DIA) has emerged as a promising strategy largely owing to developments in advanced mass spectrometers and sophisticated data analysis methods. Nevertheless, the highly complex multiplexed MS/MS spectra produced by DIA remain challenging to interpret. Here, we present a novel strategy to analyze DIA data, based on unambiguous precursor mass assignment through the mPE-MMR (multiplexed post-experimental monoisotopic mass refinement) ...

Proteogenomic Characterization of Human Early-Onset Gastric Cancer

Mun, D.-G.; Bhin, J.; Kim, S.; Kim, H.; Jung, J. H.; Jung, Y.; Jang, Y. E.; Park, J. M.; Kim, H.; Jung, Y.; Lee, H.; Bae, J.; Back, S.; Kim, S.-J.; Kim, J.; Park, H.; Li, H.; Hwang, K.-B.; Park, Y. S.; Yook, J. H.; Kim, B. S.; Kwon, S. Y.; Ryu, S. W.; Park, D. Y.; Jeon, T. Y.; Kim, D. H.; Lee, J.-H.; Han, S.-U.; Song, K. S.; Park, D.; Park, J. W.; Rodriguez, H.; Kim, J. Lee, H.; Kim, K. P.; Yang, E. G.; Kim, H. K.; Paek, E.; Lee, S.; Lee, S.-W.; Hwang, D. Cancer Cell 2019, 35, 111-124.

We report proteogenomic analysis of diffuse gastric cancers (GCs) in young populations. Phosphoproteome data elucidated signaling pathways associated with somatic mutations based on mutation-phosphorylation correlations. Moreover, correlations between mRNA and protein abundances provided potential oncogenes and tumor suppressors associated with patient survival. Furthermore, integrated clustering of mRNA, protein, phosphorylation, and N-glycosylation data identified four subtypes of diffuse GCs. Distinguishing these subtypes was possible by proteomic data. Four subtypes were associated with proliferation, immune response, metabolism, and invasion, respectively; and. ...

A mitochondrial proteome profile indicative of type 2 diabetes mellitus in skeletal muscles

Chae S.; Kim, S.-J.; Koo, Y. D.; Lee, J. H.; Kim, H.; Ahn, B. Y.; Ha, Y.-C.; Kim, Y.-H.; Jang, M. G.; Koo, K.-H.; Choi, S. H.; Lim, S.; Park, Y. J.; Jang, H .C.; Hwang, D.; Lee, S.-W.; Park, K. S. Experimental & Molecular Medicine 2018, 50:129.

The pathogenesis of type 2 diabetes mellitus (T2DM) is closely associated with mitochondrial functions in insulin-responsive tissues. The mitochondrial proteome, compared with the mitochondrial genome, which only contains 37 genes in humans, can provide more comprehensive information for thousands of mitochondrial proteins regarding T2DM-associated mitochondrial functions. However, T2DM-associated protein signatures in insulin-responsive tissues are still unclear. Here, we performed extensive proteome profiling of mitochondria from skeletal muscles in nine T2DM patients and nine nondiabetic controls. A comparison of the mitochondrial proteomes identified 335 differentially expressed proteins (DEPs) between T2DM and nondiabetic...

Multi-omics analysis identifies pathways and genes involved in diffuse-type gastric carcinogenesis induced by E-cadherin, p53, and Smad4 loss in mice

Park, J. W.; Kim, M. -S.; Voon, D. C.; Kim, S.-J.; Bae, J.; Mun, D.-G.; Ko, S.-I.; Kim, H. K.; Lee, S.-W.; Kim, D.-Y. Molecular Carcinogenesis 2018, 57, 947-954.

The molecular mechanisms underlying the pathogenesis of diffuse‐type gastric cancer (DGC) have not been adequately explored due to a scarcity of appropriate animal models. A recently developed tool well suited for this line of investigation is the Pdx‐1‐Cre;Cdh1F/+ ;Trp53F/F;Smad4F/F (pChePS ) mouse model that spontaneously develops metastatic DGC showing nearly complete E‐cadherin loss. Here, we performed a proteogenomic analysis to uncover the molecular changes induced by the concurrent targeting of E‐cadherin, p53, and Smad4 loss. The gene expression profiles of mouse DGCs and in vivo gastric phenotypes from various combinations of gene knockout demonstrated that these mutations collaborate ...

Comprehensive and sensitive proteogenomics data analysis strategy based on coplementary multi-stage database search

Madar, I. H.; Lee, Y.; Wang, X.; Ko, S.-I.; Kim, H.; Mun, D.-G.; Zhang B.; Paek, E.; Lee, S.-W. International Journal of Mass Spectrometry 2018, 427, 11-19.

Proteogenomics provide opportunities for proteomic validation of gene structures, genomic alterations and functional relevance of novel findings obtained from genomic data analysis. However, for effective proteogenomic data integration, an extensive proteome profiling, approaching the gene coverage of genomics data, is critical. Here we developed a multi-stage database search method for comprehensive proteomics data analysis to complement whole transcriptome sequencing data. The method utilizes two complementary database search engines, MS-GF+ and MODa/MODi, in tandem. The MS/MS data were first subjected to MS-GF+ database search (1st stage search) and the unidentified MS/MS data from the 1st stage search were subsequently analyzed ...