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

ABSTRACT: 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.

INSTRUMENT(S): impact HD

ORGANISM(S): Escherichia Coli Mus Musculus (mouse)

TISSUE(S): Brain, Liver, Cell Culture

SUBMITTER: Hugo M. Santos

LAB HEAD: Hugo M. Santos

PROVIDER: PXD018360 | Pride | 2020-06-08

REPOSITORIES: Pride

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			Action	DRS
	Brain_Liver_Raw.zip	Other
	E.coli_II_raw.zip	Other
	E.coli_I_raw.zip	Other
	andromeda_Brain_Liver.zip	Other
	andromeda_E.coli_I.zip	Other

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Publications

Ultrasonic-Based Filter Aided Sample Preparation as the General Method to Sample Preparation in Proteomics.

Carvalho Luís B LB Capelo-Martínez José-Luis JL Lodeiro Carlos C Wiśniewski Jacek R JR Santos Hugo M HM

Analytical chemistry 20200611 13

We propose a new high-throughput ultrafast method for large-scale proteomics approaches by speeding up the classic filter aided sample preparation protocol, FASP, from overnight to 2.5 h. Thirty-six samples can be treated in 2.5 h, and the method is scalable to 96-well plate-based pipelines. After a modification of the FASP-tube, the steps of protein reduction, protein alkylation, and protein digestion of complex proteomes are done in just 5.25 min, each one under the effects of an ultrasonic fi ...[more]

PMID: 32484334

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Formaldehyde derivatization, a Side Reaction During Filter-Aided Sample Preparation

Project description:The filter-aided sample preparation (FASP) method has been commonly used for proteomic sample preparation due to its high efficiency and convenience. Herein, we report an overlooked side reaction during FASP method, resulting in a mass increment of 12 Da. We confirmed that the side reaction is caused by formaldehyde released from the spin filter and devel-oped a pretreatment step to reduce the side reaction. We further examined spin filters from different vendors and offered suggestions for different experimental purpose. Our findings would benefit the deep screening of labeling proteomics analy-sis and offer a new direction to improve performance of filter devices used for different biological and proteomics studies.

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Proteomic Analysis of the Human Amniotic Mesenchymal Stromal Cell Secretome by integrated approaches via Filter-Aided Sample Preparation

Project description:LC-MS proteomic analysis using top-down and bottom-up combined strategies via Filter-Aided Sample Preparation (FASP) was applied to the secretome of mesenchymal stromal cells isolated from the amniotic membrane of human term placenta in the attempt to define the profile of its intact as well as digested proteome and of the biomolecules possibly involved in the immunomodulatory property exerted.

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Barley leaf sample preparation

Project description:Barley is an important cereal crop all over the world. Detailed molecular characterization of barley provides the basis for development of improved cultivars, stress and drought-resistant plants. We here present an LC-MS/MS-based proteomics study of barley leaf aimed at optimization of methods to achieve efficient and unbiased trypsin digestion of proteins prior to LC-MS/MS based sequencing and quantification of peptides. We evaluated two spin filter-aided sample preparation protocols using either sodium dodecyl-sulphate (SDS) or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation. The proteomics workflow identified up to 1800 barley proteins based on sequencing of up to 7700 peptides per sample. The two spin filter-based protocols provided a 17-38% higher efficiency than the ISD protocols, including more proteins of low abundance. Among the ISD protocols, a simple one-step reduction and S-alkylation method (OP-ISD) was the most efficient for barley leaf sample preparation; it identified and quantified 1500 proteins and displayed higher peptide-to-protein inference ratio and higher average amino acid sequence coverage of proteins. The two spin filter-aided sample preparation protocols are compatible with TMT labeling for quantitative proteomics studies. They exhibited complementary performance as about 30% of the proteins were identified by either one or the other protocol, but also demonstrated a positive bias for membrane proteins when using SDC as detergent. We provide detailed protocols for efficient barley protein sample preparation for LC-MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of barley leaf samples for MS-based proteomics, however, a simple protocol provides comparable results although with different peptide digestion profile.

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S-Trap is an ultrafast sample preparation approach for shotgun proteomics

Project description:The success of shotgun proteomic analysis depends largely on how samples are prepared. Current approaches such as gel-, solution- or filter-based, although being extensively employed in the field, are time-consuming and less effective with respect to the repetitive sample processing, recovery, and overall yield. As an alternative, suspension trapping (S-Trap) filter is commercially available very recently in the format of single or 96-well filter plate. In contrast to conventional filter aided sample preparation (FASP) approach, which utilizes a molecular weight cutoff (MWCO) membrane as the filter and requires hours of processing before digestion-ready proteins can be obtained, S-Trap employs a three-dimensional porous material as filter media, and traps particulate protein suspension with subsequent depletion of interfering substances and in-filter digestion. Due to the large (sub-micron) pore size, each centrifugation cycle of S-Trap filter only takes around 1 minute, which significantly reduces the total processing time from approximately 3 hours by FASP to less than 15 minutes, suggesting an ultrafast sample preparation approach for shotgun proteomics. Here, for the first time, we comprehensively evaluate the performance of individual S-Trap filter and 96-well filter plate in the context of global protein identification and quantitation using whole cell lysate and clinically relevant sputum samples.

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

2020-04-29 | PXD018066 | 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)).

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