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Human placental tissue LC-MS/MS

ABSTRACT: This project aimed at missing protein identification from human placental tissue, combining sample preparation and mass spectrometry technology development.For sample preparation, low molecular proteins were enriched by C18 solid phase extraction, SDS-PAGE and MWCO filter. For LC-MS/MS analysis, micro-HPLC fractionation was carried out coupled with LUMOS instrument. Proteome Discoverer 2.2 software was used for data analysis to identify missing proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Siqi Liu

PROVIDER: MSV000086385 | MassIVE | Thu Oct 29 17:34:00 GMT 2020

SECONDARY ACCESSION(S): PXD014083

REPOSITORIES: MassIVE

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Publications

Alternative Strategy To Explore Missing Proteins with Low Molecular Weight.

Lin Zhilong Z Zhang Yuanliang Y Pan Huozhen H Hao Piliang P Li Siqi S He Yanbin Y Yang Huanming H Liu Siqi S Ren Yan Y

Journal of proteome research 20191108 12

Identifying more missing proteins (MPs) is an important mission of C-HPP. With the number of identified MPs being attenuated year by year (2,949 to 2,129 MPs from 2016 to 2019), we have realized that the difficulty of exploring the remaining MPs is a challenge in technique. Herein, we propose a comprehensive strategy to effectively enrich, separate, and identify proteins with low molecular weights, aiming at the discovery of MPs. Basically, a protein extract from human placenta was passed throug ...[more]

PMID: 31592669

Similar Datasets

Human placental tissue LC-MS/MS

Project description:This project aimed at missing protein identification from human placental tissue, combining sample preparation and mass spectrometry technology development.For sample preparation, low molecular proteins were enriched by C18 solid phase extraction, SDS-PAGE and MWCO filter. For LC-MS/MS analysis, micro-HPLC fractionation was carried out coupled with LUMOS instrument. Proteome Discoverer 2.2 software was used for data analysis to identify missing proteins.

2019-10-10 | PXD014083 | Pride

Simple and efficient micro-SPE tip-based sample preparation workflow to enable sensitive proteomic profiling of limited samples

Project description:In-depth LC-MS-based proteomic profiling of limited samples has been problematic due, in large part, to the inefficiency of sample preparation and attendant sample losses. To address this issue, we developed On-Micro Solid-phase Extraction Tip-based (OmSET) sample preparation for limited biological samples. OmSET is simple, efficient, reproducible, and scalable, and is a widely accessible method for processing ~200 to 10,000 cells. The developed method benefits from minimal sample processing volumes (1-3 μL) and conducting all sample processing steps on-membrane within a single microreactor. Here, we assessed the feasibility of using micro-SPE tips for nanogram-level amounts of tryptic peptides, minimized the number of required sample handling steps and reduced the hands-on time, and evaluated the capability of OmSET for quantitative analysis of low numbers of human monocytes.

2021-02-23 | PXD022313 | Pride

Enhanced Quantitative LC-MS/MS Analysis of N-linked Glycans Derived from Glycoproteins Using Sodium Deoxycholate Detergent

Project description:Protein glycosylation is a common protein post-translational modification (PTM) in living organisms and has been shown to associate with multiple diseases, and thus may potentially be a biomarker of such diseases. Efficient protein/glycoprotein extraction is a key step in the preparation of N-glycans derived from glycoproteins prior to LC-MS analysis. Convenient, efficient and unbiased sample preparation protocols are needed. Herein, we evaluated the use of sodium deoxycholate (SDC) acidic labile detergent to release N-glycans of glycoproteins derived from biological samples such as cancer cell lines. Compared to the filter aided sample preparation approach, the sodium deoxycholate (SDC) assisted approach was determined to be more efficient and unbiased. SDC removal was determined to be more efficient when using acidic precipitation rather than ethyl acetate phase transfer. Efficient extraction of proteins/glycoproteins from biological samples was achieved by combining SDC lysis buffer and beads beating cell disruption. This was suggested by a significant overall increase in the intensities of N-glycans released from cancer cell lines. Additionally, the use of SDC approach was also shown to be more reproducible than those methods that do not use SDC.

2018-05-16 | PXD008687 | Pride

S-Trap eliminates Pluronic F68 and cell culture media polymers for effective proteomic analysis of mammalian cell bioreactor supernatants

Project description:Proteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during sample preparation. Here, we compared four different sample preparation methods to eliminate Pluronic F68 from filtered bioreactor supernatant samples: organic solvent precipitation; filter-assisted sample preparation (FASP); S-Trap; and single-pot, solid-phase, sample preparation (SP3). We found that SP3 and S-Trap substantially reduced or eliminated the polymer(s), but S-Trap provided the most robust clean-up and highest quality data. Additionally, we observed that SP3 sample preparation of our samples and in other published datasets was associated with partial alkylation of cysteines, which could impact the confidence and robustness of protein identification and quantification. Finally, we observed that several commercial mammalian cell culture media and media supplements also contained polymers with a similar mass spectrometry profile to Pluronic F68, and we suggest that proteomic analyses in these media will also benefit from the use of S-Trap sample preparation.

2020-06-30 | PXD017214 | Pride

HowDirty: An R package to evaluate molecular contaminants in LC-MS experiments

Project description:Contaminants derived from consumables, reagents, and sample handling often negatively affect LC-MS data acquisition. In proteomics experiments, they can markedly reduce identification performance, reproducibility, and quantitative robustness. Here, we introduce a data analysis workflow combining MS1 feature extraction in Skyline with HowDirty, an R-markdown-based tool, that automatically generates an interactive report on the molecular contaminant level in LC-MS data sets. To facilitate the interpretation of the results, the HTML report is self-contained and self-explanatory, including plots that can be easily interpreted. The R package HowDirty is available from https://github.com/DavidGZ1/HowDirty. To demonstrate a showcase scenario for the application of HowDirty, we assessed the impact of ultrafiltration units from different providers on sample purity after filter-assisted sample preparation (FASP) digestion. This allowed us to select the filter units with the lowest contamination risk. Notably, the filter units with the lowest contaminant levels showed higher reproducibility regarding the number of peptides and proteins identified. Overall, HowDirty enables the efficient evaluation of sample quality covering a wide range of common contaminant groups that typically impair LC-MS analyses, facilitating taking corrective or preventive actions to minimize instrument downtime.

2023-10-12 | PXD044032 | JPOST Repository

Ultrasonic-based filter aided sample preparation as the general method to sample preparation in proteomics

Project description:In this work, we propose a new high-throughput ultrafast method for large scale proteomics approaches by speeding the classic filter aided sample preparation protocol, FASP. The new US-FASP method matches the analytical minimalism roles as time, cost, sample requirement, reagent consumption, energy requirements and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all the advantages of the filter aided sample preparation protocol are maintained.

2020-06-08 | PXD018360 | Pride

GASP and FASP are complementary for LC-MS/MS proteomic analysis of drug-metabolising enzymes and transporters in pig liver

Project description:Sample preparation is a critical step in the proteomic workflow. Numerous different approaches are used, tailored to the type of sample, the aims of the experiment, analytical method, and to an extent, user preference. This has resulted in large variation in reported protein abundances. In this study we demonstrate the complementarity of two different sample preparation techniques for the study of absorption, distribution, metabolism and excretion (ADME) related proteins from pig liver tissue. Filter-aided sample preparation (FASP) is a well-established and widely used method, whilst gel-aided sample preparation (GASP) is a relatively new method optimised and simplified from previous gel-associated digestion techniques. To investigate each method the number of peptides and proteins characterised, reproducibility of results and their real-time application were examined. Whilst both methods have their merits and limitations, for example, FASP is the less technical of the two methods, whilst GASP is time efficient, ultimately the two methods show significant differences in the peptides identified and therefore the use of both methods should be considered when examining and quantifying ADME related proteins.

2018-11-12 | PXD011324 | Pride

On-chip sample preparation using a ChipFilter coupled to NanoLC-MS/MS for bottom-up proteomics

Project description:Sample preparation is a crucial step in bottom-up proteomics. Analytical performances of bottom-up proteomics can be improved by the miniaturization of sample preparation steps. Many microfluidic devices are proposed in the field of proteomics. But many of them are not capable of handling complex sample and do not integrate the processing and digestion steps. We propose a ChipFilter Proteolysis (CFP) microfluidic device derived from the Filter Aided Sample Preparation FASP method for the miniaturization of protein processing and digestion steps in bottom-up proteomics. The microchip has two reaction chambers of 0.6 µL volume separated by a protein filtration membrane in regenerated cellulose. Cell lysis, protein concentration and rapid chemical and enzymatic treatment can be performed in our microfluidic device. Complex proteomic samples like yeast protein extract have already been analyzed with our microchip. Compared to the traditional FASP method, our microfluidic device offers a better proteome coverage with ten times less starting material and eight times quicker protocol.

2020-04-29 | PXD018066 | Pride

On-chip sample preparation using a ChipFilter coupled to NanoLC-MS/MS for bottom-up proteomics

2020-04-29 | PXD016742 | Pride

Proteomic Sample Preparation in the Low Microgram Range – A Critical Evaluation of Current Workflows

Project description:Efficient and reproducible sample preparation is a prerequisite for any robust and sensitive quantitative bottom-up proteomics workflow. Over the past years, several protocols been specifically developed for the preparation of samples, which are limited in quantity. In the present study, we carried out an independent comparison between three recently described methods: Single-Pot Solid-Phase-enhanced Sample Preparation (SP3), Filter Aided Sample Preparation (FASP) and a commercial kit based on the in-Stage Tip (iST) method. We assessed their performance for the processing of proteomic samples in the low μg-range using varying amounts of HeLa cell lysate (1 µg - 20 μg of total protein). All three workflows showed similar performance for 20 µg of starting material. When handling sample sizes below 10 µg, the number of identified proteins and peptides as well as the quantitative reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low µg-range. Even when digesting 1 µg of starting material both methods still enabled the identification of over 3,000 proteins and between 25,000 to 30,000 peptides. On average, the quantitative reproducibility between experimental replicates was slightly higher in case of SP3 (R2= 0.97 (SP3); R2= 0.93 (iST)).

2018-05-11 | PXD006760 | Pride

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