Proteomics

Dataset Information

27

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


ABSTRACT: 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) and 25,000 murine macrophages isolated by flow cytometric cell sorting (FACS). 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)).

INSTRUMENT(S): Synapt MS

ORGANISM(S): Mus musculus  

TISSUE(S): Macrophage, Bone Marrow-derived Macrophage

DISEASE(S): Not Available

SUBMITTER: Malte Sielaff   

PROVIDER: PXD006760 | Pride | 2017-10-02

REPOSITORIES: Pride

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Publications

Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range.

Sielaff Malte M   Kuharev Jörg J   Bohn Toszka T   Hahlbrock Jennifer J   Bopp Tobias T   Tenzer Stefan S   Distler Ute U  

Journal of proteome research 20171011 11


Efficient and reproducible sample preparation is a prerequisite for any robust and sensitive quantitative bottom-up proteomics workflow. Here, we performed an independent comparison between single-pot solid-phase-enhanced sample preparation (SP3), filter-aided sample preparation (FASP), and a commercial kit based on the in-StageTip (iST) method. We assessed their performance for the processing of proteomic samples in the low μg range using varying amounts of HeLa cell lysate (1-20 μg of total pr  ...[more]

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