Quantitative shotgun proteome analysis by direct infusion.
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ABSTRACT:
SUBMITTER: Meyer JG
PROVIDER: S-EPMC8009190 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Large-scale proteome analysis requires rapid and high-throughput analytical methods. We recently reported a new paradigm in proteome analysis where direct infusion and ion mobility are used instead of liquid chromatography (LC) to achieve rapid and high-throughput proteome analysis. Here, we introduce an improved direct infusion shotgun proteome analysis protocol including label-free quantification (DISPA-LFQ) using CsoDIAq software. With CsoDIAq analysis of DISPA data, we can now identify up to ∼2000 proteins from the HeLa and 293T proteomes, and with DISPA-LFQ, we can quantify ∼1000 proteins from no more than 1 μg of sample within minutes. The identified proteins are involved in numerous valuable pathways including central carbon metabolism, nucleic acid replication and transport, protein synthesis, and endocytosis. Together with a high-throughput sample preparation method in a 96-well plate, we further demonstrate the utility of this technology for performing high-throughput drug analysis in human 293T cells. The total time for data collection from a whole 96-well plate is approximately 8 h. We conclude that the DISPA-LFQ strategy presents a valuable tool for fast identification and quantification of proteins in complex mixtures, which will power a high-throughput proteomic era of drug screening, biomarker discovery, and clinical analysis.
| S-EPMC9850400 | biostudies-literature
Project description:Barley seed proteins are of prime importance to the brewing industry, human and animal nutrition and in plant breeding for cultivar identification. To obtain comprehensive proteomic data from seeds, total protein from a two-rowed (Conrad) and a six-rowed (Lacey) barley cultivar were precipitated in acetone, digested in-solution, and the resulting peptides were analyzed by nano-liquid chromatography coupled with tandem mass spectrometry.The raw mass spectra data searched against Uniprot's Barley database using in-house Mascot search engine identified 1168 unique proteins. Gene Ontology (GO) analysis indicated that the majority of the seed proteins were cytosolic, with catalytic activity and associated with carbohydrate metabolism. Spectral counting analysis showed that there are 20 differentially abundant seed proteins between the two-rowed Conrad and six-rowed Lacey cultivars.This study paves the way for the use of a top-down gel-free proteomics strategy in barley for investigating more complex traits such as malting quality. Differential abundance of hordoindoline proteins impact the seed hardness trait of barley cultivars.
| S-EPMC5219712 | biostudies-literature
Project description:A major scientific challenge at the present time for cancer research is the determination of the underlying biological basis for cancer development. It is further complicated by the heterogeneity of cancer's origin. Understanding the molecular basis of cancer requires studying the dynamic and spatial interactions among proteins in cells, signaling events among cancer cells, and interactions between the cancer cells and the tumor microenvironment. Recently, it has been proposed that large-scale protein expression analysis of cancer cell proteomes promises to be valuable for investigating mechanisms of cancer transformation. Advances in mass spectrometry technologies and bioinformatics tools provide a tremendous opportunity to qualitatively and quantitatively interrogate dynamic protein-protein interactions and differential regulation of cellular signaling pathways associated with tumor development. In this review, progress in shotgun proteomics technologies for examining the molecular basis of cancer development will be presented and discussed.
| S-EPMC2352161 | biostudies-other
Project description:Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided approximately 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.
| S-EPMC2650121 | biostudies-literature
Project description:BackgroundAlthough a great deal of rice proteomic research has been conducted, there are relatively few studies specifically addressing the rice grain proteome. The existing rice grain proteomic researches have focused on the identification of differentially expressed proteins or monitoring protein expression patterns during grain filling stages.ResultsProteins were extracted from rice grains 10, 20, and 30 days after flowering, as well as from fully mature grains. By merging all of the identified proteins in this study, we identified 4,172 non-redundant proteins with a wide range of molecular weights (from 5.2 kDa to 611 kDa) and pI values (from pH 2.9 to pH 12.6). A Genome Ontology category enrichment analysis for the 4,172 proteins revealed that 52 categories were enriched, including the carbohydrate metabolic process, transport, localization, lipid metabolic process, and secondary metabolic process. The relative abundances of the 1,784 reproducibly identified proteins were compared to detect 484 differentially expressed proteins during rice grain development. Clustering analysis and Genome Ontology category enrichment analysis revealed that proteins involved in the metabolic process were enriched through all stages of development, suggesting that proteome changes occurred even in the desiccation phase. Interestingly, enrichments of proteins involved in protein folding were detected in the desiccation phase and in fully mature grain.ConclusionThis is the first report conducting comprehensive identification of rice grain proteins. With a label free shotgun proteomic approach, we identified large number of rice grain proteins and compared the expression patterns of reproducibly identified proteins during rice grain development. Clustering analysis, Genome Ontology category enrichment analysis, and the analysis of composite expression profiles revealed dynamic changes of metabolisms during rice grain development. Interestingly, we detected that proteins involved in glycolysis, TCA-cycle, lipid metabolism, and proteolysis accumulated at higher levels in fully mature grain compared to grain developing stages, suggesting that the accumulation of these proteins during the desiccation stage may be associated with the preparation of proteins required in germination.
| S-EPMC3190340 | biostudies-literature
Project description:Escherichia coli (strain ATCC 25922 in a stationary culture) cells were lysed with SDS and the lysates were processed according MED-FASP protocol. The released peptides were analyzed by LC-MS/MS. Protein content per bacterial cell was calculated on the basis of the DNA content. Absolute protein quantitation was performed using the 'Total Protein Approach'. The data are supplied in the article.
| S-EPMC4459560 | biostudies-literature
Project description:Rapid Profiling of 2000 Human Protein Groups with Direct Infusion Shotgun Proteome Analysis (DISPA) + CsoDIAq Software
2022-05-25 | MSV000089546 | MassIVE
Project description:Filter aided sample preparation (FASP) is becoming a central method for proteomic sample cleanup and peptide generation prior to LC-MS analysis. We previously adapted this method to a 96-well filter plate, and applied to prepare protein digests from cell lysate and body fluid samples in a high throughput quantitative manner. While the 96FASP approach is scalable and can handle multiple samples simultaneously, two key advantages compared to single FASP, it is also time-consuming. The centrifugation-based liquid transfer on the filter plate takes 3-5 times longer than single filter. To address this limitation, we now present a quick 96FASP (named q96FASP) approach that, relying on the use of filter membranes with a large MWCO size (~30kDa), significantly reduces centrifugal times. We show that q96FASP allows the generation of protein digests derived from whole cell lysates and body fluids in a quality similar to that of the single FASP method. Processing a sample in multiple wells in parallel, we observed excellent experimental repeatability by label-free quantitation approach. We conclude that the q96FASP approach promises to be a promising cost- and time-effective method for shotgun proteomics and will be particularly useful in large scale biomarker discovery studies.SignificanceHigh throughput sample processing is of particular interests for quantitative proteomics. The previously developed 96FASP is high throughput and appealing, however it is time-consuming in the context of centrifugation-based liquid transfer (~1.5h per spin). This study presents a truly high throughput sample preparation method based on large cut-off 96-well filter plate, which shortens the spin time to ~20min. To our knowledge, this is the first multi-well method that is entirely comparable with conventional FASP. This study thoroughly examined two types of filter plates and performed side-by-side comparisons with single FASP. Two types of samples, whole cell lysate of a UTI (urinary tract infection)-associated Klebsiella pneumoniae cell and human urine, were tested which demonstrated its capability for quantitative proteomics. The q96FSAP approach makes the filter plate-based approach more appealing for protein biomarker discovery projects, and could be broadly applied to large scale proteomics analysis.
| S-EPMC6029622 | biostudies-literature
Project description:BackgroundProteogenomics aims to utilize experimental proteome information for refinement of genome annotation. Since mass spectrometry-based shotgun proteomics approaches provide large-scale peptide sequencing data with high throughput, a data repository for shotgun proteogenomics would represent a valuable source of gene expression evidence at the translational level for genome re-annotation.DescriptionHere, we present OryzaPG-DB, a rice proteome database based on shotgun proteogenomics, which incorporates the genomic features of experimental shotgun proteomics data. This version of the database was created from the results of 27 nanoLC-MS/MS runs on a hybrid ion trap-orbitrap mass spectrometer, which offers high accuracy for analyzing tryptic digests from undifferentiated cultured rice cells. Peptides were identified by searching the product ion spectra against the protein, cDNA, transcript and genome databases from Michigan State University, and were mapped to the rice genome. Approximately 3200 genes were covered by these peptides and 40 of them contained novel genomic features. Users can search, download or navigate the database per chromosome, gene, protein, cDNA or transcript and download the updated annotations in standard GFF3 format, with visualization in PNG format. In addition, the database scheme of OryzaPG was designed to be generic and can be reused to host similar proteogenomic information for other species. OryzaPG is the first proteogenomics-based database of the rice proteome, providing peptide-based expression profiles, together with the corresponding genomic origin, including the annotation of novelty for each peptide.ConclusionsThe OryzaPG database was constructed and is freely available at http://oryzapg.iab.keio.ac.jp/.
| S-EPMC3094275 | biostudies-literature
Project description:PurposeThe purpose of this investigation was to better understand lipid composition in human meibum.MethodsIntact lipids in meibum samples were detected by direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis in positive detection mode using sodium iodide (NaI) as an additive. The peak intensities of all major types of lipid species, that is, wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs) were corrected for peak overlapping and isotopic distribution; an additional ionization efficiency correction was performed for WEs and CEs, which was simplified by the observation that the corresponding ionization efficiency was primarily dependent on the specific lipid class and saturation degree of the lipids while independent of the carbon chain length. A set of WE and CE standards was spiked in meibum samples for ionization efficiency determination and absolute quantitation.ResultsThe absolute amount (μmol/mg) for each of 51 WEs and 31 CEs in meibum samples was determined. The summed masses for 51 WEs and 31 CEs accounted for 48 ± 4% and 40 ± 2%, respectively, of the total meibum lipids. The mass percentages of saturated and unsaturated species were determined to be 75 ± 2% and 25 ± 1% for CEs and 14 ± 1% and 86 ± 1% for WEs. The profiles for two types of DEs were also obtained, which include 42 α,ω Type II DEs, and 21 ω Type I-St DEs.ConclusionsMajor neutral lipid classes in meibum samples were quantitatively profiled by ESI-MS analysis with NaI additive.
| S-EPMC3753034 | biostudies-literature