Dataset Information

NIST Candidate RM 8461 Human Liver for Proteomics

ABSTRACT: Advances in mass spectrometry (MS) instrumentation resulting in higher mass resolution and higher duty cycles have allowed deeper coverages of proteomes. The development of candidate RM 8461 Human Liver for Proteomics was undertaken to provide the community with a sample with suitable complexity for method development and validation, instrument and data validation, and determining identification QC thresholds. Herein we describe a comparison of methods and homogeneity assessment in which candidate RM 8461 Human Liver for Proteomics is solubilized, the proteins reduced, alkylated, digested and the tryptic peptides and their modifications are identified by bottom up LC/MS/MS to assess deep (e.g. thousands of identifications) protein coverage.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Liver

SUBMITTER: W. Clay Davis

LAB HEAD: Clay Davis

PROVIDER: PXD013608 | Pride | 2020-11-20

REPOSITORIES: Pride

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Publications

Characterization of a human liver reference material fit for proteomics applications.

Davis W Clay WC Kilpatrick Lisa E LE Ellisor Debra L DL Neely Benjamin A BA

Scientific data 20191218 1

The National Institute of Standards and Technology (NIST) is creating new, economical, qualitative reference materials and data for proteomics comparisons, benchmarking and harmonization. Here we describe a large dataset from shotgun proteomic analysis of RM 8461 Human Liver for Proteomics, a reference material being developed. Consensus identifications using multiple search engines and sample preparations demonstrate a homogeneous and fit-for-purpose material that can be incorporated into autom ...[more]

PMID: 31852895

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Project description:Recent advances in mass spectrometric (MS) instruments resulting in high mass resolution and high duty cycles have allowed of very deep coverage of proteomes. However, even with state of the art, high duty cycle instruments, the number of proteins identified with a conventional single liquid chromatography-tandem MS (LC/MS/MS) analysis is typically limited and fractionating protein samples prior to LC/MS/MS analysis is crucial for increasing both the analytical dynamic range and proteome coverage. In order to achieve near comprehensive identification of proteomes two-dimensional (2D) chromatography has been an invaluable tool. This first dimension is typically performed with µL/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range while the second dimension is typically performed with nL/min flow rates and capillary columns with small inner diameters. Typically, reverse phase liquid chromatography (RPLC) provides high peak resolution of peptides and achieves higher peak capacities than strong cation exchange chromatography (SCX) due to the faster chromatographic partitioning. Another advantage of RPLC is that the mobile phases used generate much cleaner samples for downstream analyses, while the incorporation of a desalting step for SCX in order to eliminated the high salt concentrations that can significantly decrease the analytical sensitivity of the MS analysis due to ionization problems. When operated at widely different pH values (e.g., 1st dimension pH 10 and 2nd dimension pH 3) 2D RPLC-RPLC provides separation orthogonality comparable to that of the combination of SCX-RPLC. The difference of separation selectivity between the low and high pH (HpH) RPLC comes from the changes in the charge distribution within peptide chains upon altering pH of the eluent. Herein we describe a method in which tryptic peptides are separated in the first dimension by HpH-RPLC and fractions are collected every 90 s over an 80 min gradient. For the second dimension, each of these fractions is individually run by the standard low pH RPLC method. The implementation of the HpH pre-fractionation allows for short second dimension gradients for bottom up LC/MS/MS and yields very deep (e.g. thousands of identifications) protein coverage at reasonable measurement time.

Project description:Background: Glioblastoma is the most aggressive form of brain tumors showing resistance to treatment with various chemotherapeutic agents. The most effective way to eradicate glioblastoma requires the concurrent inhibition of multiple signaling pathways and target molecules involved in the progression of glioblastoma. Recently, we obtained a series of 1,2,3,4-tetrahydroisoquinoline alkaloids with potent anti-cancer activities, including ecteinascidin-770 (ET-770; the compound 1a) and renieramycin M (RM; the compound 2a) from Thai marine invertebrates, together with a 2’-N-4”-pyridinecarbonyl derivative of ET-770 (ET-770-DR; the compound 3). We attempted to characterize the molecular pathways responsible for cytotoxic effects of these compounds on a human glioblastoma cell line U373MG. Methods: We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. Results: All of these compounds dissolved in dimethyl sulfoxide (DMSO) as a vehicle induced apoptosis of U373MG cells at nanomolar concentrations. The compound 3 reduced the expression of 417 genes and elevated the levels of 84 genes, while ET-770 downregulated 426 genes and upregulated 45 genes. RM decreased the expression of 274 genes and increased the expression of 9 genes. The set of 196 downregulated genes and 6 upregulated genes showed an overlap among all the compounds, suggesting an existence of the common pathways involved in induction of apoptosis. We identified the ErbB (EGFR) signaling pathway as one of the common pathways enriched in the set of downregulated genes, composed of PTK2, AKT3, and GSK3B serving as key molecules that regulate cell movement and the nervous system development. Furthermore, a GSK3B-specific inhibitor induced apoptosis of U373MG cells, supporting an anti-apoptotic role of GSK3B. Conclusion: Molecular network analysis is a useful approach not only to characterize the glioma-relevant pathways but also to identify the network-based effective drug targets. To determine the 50% inhibitory concentration (IC50), U373MG human glioblastoma cells, incubated in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin (feeding medium), were exposed to the chemical compounds for varying periods at variable concentrations. Then, we assessed the cell viability by morphological observations and by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell growth kit (Millipore). The cells were incubated for 4 to 72 hours in the feeding medium with inclusion of the chemical compounds at the IC50 concentration or the vehicle (DMSO) at an equivalent concentration (v/v), and then were processed for microarray analysis at 24 hours after exposure and western blot analysis of the cleavage of poly-ADP-ribose-polymerase (PARP) and caspase-3 at 4 to 72 hours after exposure.

Dataset Information

NIST Candidate RM 8461 Human Liver for Proteomics

Publications

Characterization of a human liver reference material fit for proteomics applications.

Similar Datasets

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