Universal Pretreatment Development for Low-input Proteomics Using Lauryl Maltose Neopentyl Glycol.

In recent years, there has been a growing demand for low-input proteomics, particularly in the context of single-cell proteomics (SCP). In this study, we have developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method. This method effectively reduces protein and peptide loss in samples by incorporating LMNG, a surfactant, into the digestion solution and subsequently removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is the prevention of mass spectrometry (MS) contamination. When we applied the LASP method to the low-input SP3 method and on-bead digestion in coimmunoprecipitation-MS, we observed a significant improvement in the recovery of the digested peptides. Furthermore, we have established a simple and easy sample preparation method for SCP based on the LASP method and identified a median of 1175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.

GeLC-FAIMS-MS workflow for in-depth middle-down proteomics.

Middle-down proteomics (MDP) is an analytical approach in which protein samples are digested with proteases such as Glu-C to generate large peptides (>3 kDa) that are analyzed by mass spectrometry (MS). This method is useful for characterizing high-molecular-weight proteins that are difficult to detect by top-down proteomics (TDP), in which intact proteins are analyzed by MS. In this study, we applied GeLC-FAIMS-MS, a multidimensional separation workflow that combines gel-based prefractionation with LC-FAIMS MS, for deep MDP. Middle-down peptides generated by optimized limited Glu-C digestion conditions were first size-fractionated by polyacrylamide gel electrophoresis, followed by C4 reversed-phase liquid chromatography separation and additional ion mobility fractionation, resulting in a significant increase in peptide length detectable by MS. In addition to global analysis, the GeLC-FAIMS-MS concept can also be applied to targeted MDP, where only proteins in the desired molecular weight range are gel-fractionated and their Glu-C digestion products are analyzed, as demonstrated by targeted analysis of integrins in exosomes. In-depth MDP achieved by global and targeted GeLC-FAIMS-MS supports the exploration of proteoform information not covered by conventional TDP by increasing the number of detectable protein groups or post-translational modifications (PTMs) and improving the sequence coverage.

Nucleobindin-2 Mediates Transforming Growth Factor-ß1-Driven Phenotypes in Zinc Finger E-Box Binding Homeobox 1-High Uterine Carcinosarcoma.

Epithelial-mesenchymal transition is a hallmark of uterine carcinosarcoma (UCS). Here, shotgun proteomics analysis used to identify biomarkers associated with blebbistatin-mediated epithelial-mesenchymal transition in UCS indicated up-regulation of nucleobindin-2 (NUCB2) in endometrial carcinoma (Em Ca) cells. Expression of N-cadherin, Snail, Slug, and ZEB1 was reduced in NUCB2 knockout Em Ca cells, whereas ZEB1, Twist1, and vimentin were up-regulated in NUCB2-overexpressing Em Ca cells. NUCB2 knockout reduced cell proliferation and migration, whereas NUCB2 overexpression had the opposite effect. Treatment of Em Ca cells with transforming growth factor (TGF)-ß1 dramatically altered morphology toward a fibroblastic appearance; concomitantly, expression of NUCB2 and ZEB1 increased. The NUCB2 promoter was also activated by transfection of Smad2. In UCS tissues, NUCB2 expression was significantly higher in sarcomatous compared with carcinomatous components, which was consistent with increased TGF-ß1 mRNA expression in stromal and sarcomatous components compared with carcinomatous components. In addition, NUCB2 score correlated positively with ZEB1 and vimentin scores, whereas ZEB1 score correlated positively with Slug and vimentin scores and inversely with the E-cadherin score. Collectively, these data indicate that TGF-ß-dependent up-regulation of NUCB2 and ZEB1 contributes to the phenotypic characteristics of sarcomatous components in UCS.

Denonvilliers' fascia as a potential nerve-course marker for the female urinary bladder.

OBJECTIVES: This study investigated the relationship between Denonvilliers' fascia (DF) and the pelvic plexus branches in women and explored the possibility of using the DF as a positional marker in nerve-sparing radical hysterectomy (RH). METHODS: This study included eight female cadavers. The DF, its lateral border, and the pelvic autonomic nerves running lateral to the DF were dissected and examined. The pelvis was cut into two along the mid-sagittal line. The uterine artery, deep uterine veins, vesical veins, and nerve branches to the pelvic organs were carefully dissected. RESULTS: The nerves ran sagitally, while the DF ran perpendicularly to them. The rectovaginal ligament was continuous with the DF, forming a single structure. The DF attached perpendicularly and seamlessly to the pelvic plexus. The pelvic plexus branches were classified into a ventral part branching to the bladder, uterus, and upper vagina and a dorsal part branching to the lower vagina and rectum as well as into four courses. Nerves were attached to the rectovaginal ligament and ran on its surface to the bladder ventral to the DF. The uterine branches split from the common trunk of these nerves. The most dorsal branch to the bladder primarily had a common trunk with the uterine branch, which is the most important and should be preserved in nerve-sparing Okabayashi RH. CONCLUSION: The DF can be used as a marker for nerve course, particularly in one of the bladder branches running directly superior to the DF, which can be preserved in nerve-sparing Okabayashi RH.

Proteome Analysis of Serum Purified Using Solanum tuberosum and Lycopersicon esculentum Lectins.

Serum and plasma exhibit a broad dynamic range of protein concentrations, posing challenges for proteome analysis. Various technologies have been developed to reduce this complexity, including high-abundance depletion methods utilizing antibody columns, extracellular vesicle enrichment techniques, and trace protein enrichment using nanobead cocktails. Here, we employed lectins to address this, thereby extending the scope of biomarker discovery in serum or plasma using a novel approach. We enriched serum proteins using 37 different lectins and subjected them to LC-MS/MS analysis with data-independent acquisition. Solanum tuberosum lectin (STL) and Lycopersicon esculentum lectin (LEL) enabled the detection of more serum proteins than the other lectins. STL and LEL bind to N-acetylglucosamine oligomers, emphasizing the significance of capturing these oligomer-binding proteins when analyzing serum trace proteins. Combining STL and LEL proved more effective than using them separately, allowing us to identify over 3000 proteins from serum through single-shot proteome analysis. We applied the STL/LEL trace-protein enrichment method to the sera of systemic lupus erythematosus model mice. This revealed differences in >1300 proteins between the systemic lupus erythematosus model and control mouse sera, underscoring the utility of this method for biomarker discovery.

Development of a Simple and Stable NanoESI Spray System Using Suction Wind from the MS Inlet.

Improving the reproducibility of proteome analysis systems presents a challenge; therefore, in this study, we developed a new insertion spray ionization (InSpIon) system wherein an InSpIon tube was designed with a spray tip inserted into the tube. This system stabilized the spray and subsequently improved the reproducibility of the analysis.

In-depth proteomic analysis of juvenile dermatomyositis serum reveals protein expression associated with muscle-specific autoantibodies.

OBJECTIVES: The clinical symptoms and complications of JDM differ depending on the type of muscle-specific autoantibodies (MSAs) present. We aimed to identify protein expression profiles specific for MSAs that characterize various clinical features by comprehensively analyzing the proteins present in the serum of patients with JDM. METHODS: We analysed sera from patients with JDM that were positive for anti-melanoma differentiation-associated protein 5 (MDA5) antibodies (n = 5), anti-nuclear matrix protein 2 (NXP2) antibodies (n = 5) and anti-transcriptional intermediary factor 1 alpha or gamma subunit (TIF1-γ) antibodies (n = 5), and evaluated healthy controls (n = 5) via single-shot liquid chromatography-tandem mass spectrometry (MS) in data-independent acquisition mode, which is superior for comparative quantitative analysis. We identified different protein groups based on MSAs and performed pathway analysis to understand their characteristics. RESULTS: We detected 2413 proteins from serum MS analysis; 508 proteins were commonly altered in MSAs, including many myogenic enzymes and IFN-regulated proteins. Pathway analysis using the top 50 proteins that were upregulated in each MSA group revealed that the type I IFN and proteasome pathways were significantly upregulated in the anti-MDA5 antibody group alone. CONCLUSION: Although JDM serum contains many proteins commonly altered in MSAs, the pathways associated with clinical features of MSAs differ based on protein accumulation. In-depth serum protein profiles associated with MSAs may be useful for developing therapeutic target molecules and biomarkers.

Clinical and Sociodemographic Factors Associated With Health-Related Quality of Life in Patients With Adult Congenital Heart Disease　- A Nationwide Cross-Sectional Multicenter Study.

BACKGROUND: Little is known about clinical or sociodemographic factors that influence health-related quality of life (HRQoL) in patients with adult congenital heart disease (ACHD).Methods and Results: We conducted a nationwide prospective cross-sectional multicenter study at 4 large ACHD centers in Japan. From November 2016 to June 2018, we enrolled 1,223 ACHD patients; 1,025 patients had an HRQoL score. Patients completed a questionnaire survey, including sociodemographic characteristics, and the 36-Item Short-Form Health Survey (SF-36). To determine factors associated with HRQoL, correlations between 2 SF-36 summary scores (i.e., physical component score [PCS] and mental component score [MCS]) and other clinical or sociodemographic variables were examined using linear regression analysis. In multivariable analysis, poorer PCS was significantly associated with 11 variables, including older age, higher New York Heart Association class, previous cerebral infarction, being unemployed, and limited participation in physical education classes and sports clubs. Poorer MCS was associated with congenital heart disease of great complexity, being part of a non-sports club, current smoking, and social drinking. Student status and a higher number of family members were positively correlated with MCS. CONCLUSIONS: This study demonstrates that HRQoL in ACHD patients is associated with various clinical and sociodemographic factors. Further studies are needed to clarify whether some of these factors could be targets for future intervention programs to improve HRQoL outcomes.

Evaluation of the Suitability of Dried Saliva Spots for In-Depth Proteome Analyses for Clinical Applications.

Previously, we performed nontargeted proteome analysis using dried blood spots (DBSs) that are widely used in newborn screening for the clinical diagnosis of congenital genetic diseases and immunodeficiency. We have developed an efficient and simple pretreatment method for DBSs that can detect more than 1000 proteins. To complement proteins that are difficult to detect via DBS analysis with less invasive alternative body fluids, we conducted this study to investigate the proteins detected from dried saliva spots (DSSs) using single-shot LC-MS/MS, which is practical in clinical settings. We also clarified whether DSSs have the same advantages as DBSs, and we investigated the influence of saliva collection conditions and the storage environment on their protein profile. As a result, we detected approximately 5000 proteins in DSSs and whole saliva, and we concluded that they were sufficient to complement the proteins lacking in the blood analysis. DSSs could be used as an alternative tool to DBSs for detecting the presence of causative proteins.

Single-Shot 10K Proteome Approach: Over 10,000 Protein Identifications by Data-Independent Acquisition-Based Single-Shot Proteomics with Ion Mobility Spectrometry.

The evolution of mass spectrometry (MS) and analytical techniques has led to the demand for proteome analysis with high proteome coverage in single-shot measurements. Focus has been placed on data-independent acquisition (DIA)-MS and ion mobility spectrometry as techniques for deep proteome analysis. We aimed to expand the proteome coverage by single-shot measurements using optimizing high-field asymmetric waveform ion mobility spectrometry parameters in DIA-MS. With our established proteome analysis system, more than 10,000 protein groups were identified from HEK293 cell digests within 120 min of MS measurement time. Additionally, we applied our approach to the analysis of host proteins in mouse feces and detected as many as 892 host protein groups (771 upregulated/121 downregulated proteins) in a mouse model of repeated social defeat stress (R-SDS) used in studying depression. Interestingly, 285 proteins elevated by R-SDS were related to mental disorders. The fecal host protein profiling by deep proteome analysis may help us understand mental illness pathologies noninvasively. Thus, our approach will be helpful for an in-depth comparison of protein expression levels for biological and medical research because it enables the analysis of highly proteome coverage in a single-shot measurement.

Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition.

Proteomics has become an increasingly important tool in medical and medicinal applications. It is necessary to improve the analytical throughput for these applications, particularly in large-scale drug screening to enable measurement of a large number of samples. In this study, we aimed to establish an ultrafast proteomic method based on 5-min gradient LC and quadrupole-Orbitrap mass spectrometer (Q-Orbitrap MS). We precisely optimized data-independent acquisition (DIA) parameters for 5-min gradient LC and reached a depth of >5000 and 4200 proteins from 1000 and 31.25 ng of HEK293T cell digest in a single-shot run, respectively. The throughput of our method enabled the measurement of approximately 80 samples/day, including sample loading, column equilibration, and wash running time. We demonstrated that our method is applicable for the screening of chemical responsivity via a cell stimulation assay. These data show that our method enables the capture of biological alterations in proteomic profiles with high sensitivity, suggesting the possibility of large-scale screening of chemical responsivity.

Impact of coronavirus disease 2019 on the number of newly diagnosed cancer patients and examinations and surgeries performed for cancer in Japan: a nationwide study.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has rapidly and dramatically influenced healthcare across Japan. However, the influence of the COVID-19 pandemic on the number of newly diagnosed cancer, surgical treatment, and diagnostic examination for cancer types have not been completely investigated all over Japan. This study aimed to analyze the number of cases before and during the COVID-19 pandemic. METHODS: This retrospective study was a survey that asked to provide the number of cases diagnosed with gastric, colorectal, lung, breast, and cervical cancer between January 2019 and December 2020. The survey was sent to tertiary healthcare hospitals, including national cancer institutions, university hospitals, and general hospitals, all over Japan. Data obtained from 105 of 486 surveyed hospitals were evaluated, and the number of cases in each quarter in 2020 was compared with that in the equivalent quarter in 2019. RESULTS: In the second quarter (Q2), significant reductions were observed in the median number of newly diagnosed cases from 2019 to 2020: gastric cancer, 26.7% (43 vs. 32, p <  0.001); colorectal cancer, 17.9% (52 vs. 40, p <  0.001); lung cancer, 12.3% (53.5 vs. 47, p <  0.001); and breast cancer, 13.1% (43 vs. 35.5, p <  0.001). A significant reduction of 11.4% (9 vs. 8, p = 0.03) was observed in the third quarter (Q3) for cervical cancer. In Q2, the number of cases decreased by 30.9% (25 vs. 15, p <  0.001) for stage I gastric cancer, by 27.3% (12 vs. 9, p <  0.001) for stage I colorectal cancer, and by 17.6% (13 vs. 10, p <  0.001) for stage II breast cancer. The magnitude of reduction was significant for the localized stages of gastric, colorectal, and breast cancer according to diagnostic examinations in Q2 and surgical and endoscopic treatment in Q3 rather than that for lung or cervical cancer. CONCLUSIONS: COVID-19 has prolonged collateral effects on cancer care, including examination, diagnosis, and surgery, with significant effects on gastric cancer, followed by colorectal, lung, breast, and cervical cancer in Japan.

Cytoplasmic EBP50 and elevated PARP1 are unfavorable prognostic factors in ovarian clear cell carcinoma.

Patients with ovarian clear cell carcinoma (OCCC) experience frequent recurrence, which is most likely due to chemoresistance. We used shotgun proteomics analysis and identified upregulation of ezrin-binding phosphoprotein 50 (EBP50) in recurrent OCCC samples. Cytoplasmic and/or nuclear (Cyt/N), but not membranous, EBP50 immunoreactivity was significantly higher in recurrent OCCC as compared with that of primary tumors. OCCC cells expressing cytoplasmic EBP50 were significantly less susceptible to cisplatin (CDDP)-induced apoptosis compared with cells expressing membranous EBP50. Abrogation of resistance following knockdown of cytoplasmic EBP50 was accompanied by decreased XIAP and BCL2, increased BAX and increased caspase-3 cleavage. We found that poly (ADP-ribose) polymerase1 (PARP1), which is involved in DNA damage detection and repair, binds to EBP50 through its PDZ1 domain. CDDP treatment of cells expressing cytoplasmic (but not membranous) EBP50 increased nuclear PARP1 expression, whereas knockdown of EBP50 cells decreased PARP1 expression and activity following CDDP treatment. Finally, OCCC patients with a combination of Cyt/N EBP50 and high PARP1 score had worst the prognosis for overall and progression-free survival. Together, our data suggest that cytoplasmic EBP50 inhibits apoptosis and promotes OCCC survival through stabilization of PARP1 activity and modulation of the XIAP/BCL2/BAX axis. This may increase the likelihood of tumor recurrence, and we therefore suggest a combined analysis for EBP50 and PARP1 may have great utility in OCCC prediction and prognosis.

Highly accurate and precise quantification strategy using stable isotope dimethyl labeling coupled with GeLC-MS/MS.

Shotgun proteomics is a powerful method for comprehensively identifying and quantifying tryptic peptides, but it is difficult to analyze proteolytic events. One-dimensional gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) enables the separation of proteolytic fragments using SDS-PAGE followed by identification using LC-MS/MS. GeLC-MS/MS is thus an excellent method for identifying fragmentation. However, the lower reproducibility of gel extraction and nano flow LC-MS/MS can produce inaccurate results in comparative analyses of protein quantification among samples. In this study, a novel GeLC-MS/MS method coupled with stable isotope dimethyl labeling was developed. In the method, a mixture of light- and heavy-labeled samples is loaded onto an SDS-PAGE gel, and proteins with different isotopes in one extracted band are quantitatively analyzed by one-shot injection. This procedure enables accurate determination of the abundance ratio of peptides between two samples, even in cases of low peptide abundance, and it is not affected by the reproducibility of the gel extraction or LC-MS procedures. Therefore, our new GeLC-MS/MS method coupled with stable isotope dimethyl labeling provides high accuracy and comprehensive peptide comparisons, enabling the detection of proteolysis events caused by disease or physiological processes.

A highly efficient method for extracting peptides from a single mouse hypothalamus.

Living organisms contain a variety of endogenous peptides that function as significant regulators of many biological processes. Endogenous peptides are typically analyzed using liquid chromatography-mass spectrometry (LC-MS). However, due to the low efficiency of peptide extraction and low abundance of peptides in a single animal, LC-MS-based peptidomics studies have not facilitated an understanding of the individual differences and tissue specificity of peptide abundance. In this study, we developed a peptide extraction method followed by nano-flow LC-MS/MS analysis. This method enabled highly efficient and reproducible peptide extraction from sub-milligram quantities of hypothalamus dissected from a single animal. Diverse bioactive and authentic peptides were detected from a sample volume equivalent to 135 µg of hypothalamus. This method may be useful for elucidating individual differences and tissue specificity, as well as for facilitating the discovery of novel bioactive peptides and biomarkers and developing peptide therapeutics.

A cystectomic technique with low risk of rupture for women with benign ovarian cyst.

The majority of patients with benign ovarian cysts undergo laparoscopic surgery using both cystectomy and stripping techniques. However, these techniques have difficulty correctly identifying cleavage planes and are prone to cyst rupture. We present a surgical cystectomy technique that correctly identifies the cleavage plane with a low risk of ovarian cyst rupture, even in patients with multicystic ovarian cysts. Cystectomy was performed using Maryland forceps with gentle open and close dissecting motions only. Both the surgeon and assistant handled the ovarian cortex and cyst wall, and soft traction between the cortex and cyst wall as far as the nearby dissection plane without grasping the cyst wall was essential. In patients with multicystic ovarian cysts, making a plane at the notch between cysts decreases the risk of cyst rupture. This technique allows the correct identification of the cleavage plane for dissection and avoids the risk of cyst rupture.Impact StatementWhat is already known on this subject? The majority of patients with benign ovarian cysts undergo laparoscopic surgery using both cystectomy and stripping techniques. These techniques have difficulty correctly identifying cleavage planes and are prone to cyst rupture.What do the results of this study add? This technique allows the correct identification of the cleavage plane for dissection and avoids the risk of cyst rupture.What are the implications of these findings for clinical practice and/or further research? Our technique might be useful for the preservation of the ovarian reserve because patients in this study had a low proportion of ovarian follicles in the surgical specimen.

S100A4/non-muscle myosin II signaling regulates epithelial-mesenchymal transition and stemness in uterine carcinosarcoma.

Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Although S100A4 is an inducer of EMT, little is known about its involvement in UCS tumorigenesis. Herein, we focused on the functional role of S100A4 during development of UCS. Expression of S100A4 and molecules associated with its function were also examined in 35 UCS cases. In endometrial carcinoma cell lines, S100A4 promoter activity and mRNA levels were significantly increased by the transfection of NF-κB/p65, independent of a putative κB-binding site in the promoter. Cells stably overexpressing S100A4 showed enhancement of CSC properties, along with decreased cell proliferation and acceleration of cell migration. These phenotypes were abrogated in S100A4-knockdown cells. A combination of S100A4 antibody-mediated co-immunoprecipitation and shotgun proteomics analysis revealed that S100A4 strongly interacted with non-muscle myosin II (NMII) heavy chains, including myosin 9 and myosin 14. Specific inhibition of NMII by blebbistatin phenocopied S100A4 overexpression and induced a fibroblast-like morphology. In clinical samples, S100A4 score was significantly higher in sarcomatous as compared with carcinomatous components of UCS, and was positively correlated with ALDH1, Slug, and vimentin scores, and inversely with Ki-67 labeling indices. These findings suggest that an S100A4/NMII-related signaling cascade may contribute to the establishment and maintenance of EMT/CSC properties, along with changes in cell proliferation and migration capability. These events may be initiated in carcinomatous components in UCS and lead to divergent sarcomatous differentiation.

Effectiveness of HPV vaccination against the development of high-grade cervical lesions in young Japanese women.

BACKGROUND: Although more than 10 years have passed since HPV vaccination was implemented, first as an interim programme (Emergent vaccine promotion programme) in November 2010, followed by incorporating into the National Immunization Programme in April, 2013 and suspended in June 2013, limited studies have investigated the HPV vaccine effectiveness against high-grade cervical lesions in Japan. METHODS: We collected the matched data of the results of cervical biopsy and history of vaccination from the Japan Cancer Society database. The subjects were women aged 20 to 29 years screened for cervical cancer between April, 2015 and March, 2017, and with information on HPV vaccination status. We estimated the relative risk of developing high-grade cervical lesions in vaccinated subjects using Poisson regression as compared to unvaccinated subjects. RESULTS: Among the 34,281 women screened, 3770 (11.0%) were vaccinated. The prevalence of CIN2+ was statistically significantly lower in the vaccinated women as compared to the unvaccinated women (Vaccine Effectiveness (VE) =76%; RR = 0.24, 95% CI:0.10-0.60). High VE against CIN3+ was also observed (91%; RR = 0.09, 95% CI:0.00-0.42). CONCLUSION: Women aged 20-29 years who received at least one dose of HPV vaccine had a significantly lower risk of high-grade cervical lesions than those not vaccinated. In Japan, HPV vaccination should be resumed in order to reduce the incidence of cervical cancer.

Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K cells, has potent insulin-releasing and extrapancreatic glucoregulatory activities. However, exogenous GIP has less potent biological effects compared with another incretin hormone, GLP-1, which limits its use for the treatment of type 2 diabetes. The fate and secretion of administered native GIP remain unclear. The aim of this study was to identify plasma binding proteins for human GIP. Fluorescent-labelled GIP was added to fresh human plasma and subjected to clear native polyacrylamide gel electrophoresis (CN-PAGE). Then fluorescent protein bands were in-gel trypsin-digested and subjected to liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, revealing the presence of albumin, immunoglobulin G (IgG) and transferrin. In contrast to GIP, the binding of fluorescent GLP-1 and glucagon to plasma protein fractions were minimal. CN-PAGE analysis of synthetic GIP incubated with human serum albumin, purified IgG or transferrin, and subsequent western blot analysis revealed that GIP binds to each of these proteins. Taken together, these results indicate that GIP readily binds to albumin, IgG and transferrin, three plasma proteins highly abundant in the human peripheral circulation. Separation of protein complexes using CN-PAGE and the identification of in-gel digested proteins by LC-MS/MS analysis provide a promising strategy to identify plasma binding proteins for bioactive peptides.