PXD006760-1

PXD006760 is an original dataset announced via ProteomeXchange.

Title	Proteomic Sample Preparation in the Low Microgram Range - A Critical Evaluation of Current Workflows
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) 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)).
HostingRepository	PRIDE
AnnounceDate	2017-10-02
AnnouncementXML	Submission_2017-10-02_04:15:04.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Malte Sielaff
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Synapt MS

Revision	Datetime	Status	ChangeLog Entry
0	2017-06-21 00:02:38	ID requested
⏵ 1	2017-10-02 04:15:05	announced
2	2018-05-12 00:31:27	announced	Updated project metadata.

Sielaff M, Kuharev J, Bohn T, Hahlbrock J, Bopp T, Tenzer S, Distler U, Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range. J Proteome Res, 16(11):4060-4072(2017) [pubmed]

curator keyword: Technical

submitter keyword: Bottom-up proteomics, sample preparation, filter-aided sample preparation (FASP), single-pot solid-phase-enhanced sample preparation (SP3), in-StageTip digestion (iST)

Stefan Tenzer
contact affiliation	Institute for Immunology, University Medical Center of Mainz, Langenbeckstr. 1, 55131 Mainz, Germany
contact email	tenzer@uni-mainz.de
lab head
Malte Sielaff
contact affiliation	Institute for Immunology University Medical Center of the Johannes Gutenberg University Mainz
contact email	malte.sielaff@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD006760
     1. Label: PRIDE project
     2. Name: Proteomic Sample Preparation in the Low Microgram Range - A Critical Evaluation of Current Workflows