Dataset Information

Development of sequential sample processing protocol on a single anion-exchange solid-phase extraction disc for bottom-up/cross-linking mass spectrometry

ABSTRACT: In this project, we developed a rapid enzymatic digestion protocol within an anion exchange StageTip named AnExSP (anion-exchange disc-assisted sequential sample preparation) and, using data-independent acquisition, analyzed its digestion performance of human cellular protein extracts (HCPE) using trypsin and trypsin/Lys-C. The outstanding effectiveness and simplicity of AnExSP in sample preparation for gel-based cross-linking mass spectrometry was also verified.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Nobuaki Takemori

PROVIDER: PXD029444 | JPOST Repository | Wed Apr 20 00:00:00 BST 2022

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	DIA_HCPE-10ug_AnExSP_18h_1.raw	Raw
	DIA_HCPE-10ug_AnExSP_18h_2.raw	Raw
	DIA_HCPE-10ug_AnExSP_4h_1.raw	Raw
	DIA_HCPE-10ug_AnExSP_4h_2.raw	Raw
	DIA_HCPE-10ug_AnExSP_4h_3.raw	Raw

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Publications

Bottom-up/cross-linking mass spectrometry <i>via</i> simplified sample processing on anion-exchange solid-phase extraction spin column.

Takemori Ayako A Kawashima Yusuke Y Takemori Nobuaki N

Chemical communications (Cambridge, England) 20220118 6

We introduce a simple single-column protein digestion method for low-microgram-level samples containing sodium dodecyl sulfate and Coomassie dye that can be completed within a few hours. ...[more]

PMID: 34897310

Similar Datasets

Project description:Proper sample preparation protocols represent a critical step for LC-MS-based proteomic study designs and influence the speed, performance and automation of high-throughput data acquisition. The main objective of this study was to compare two commercial solid phase extraction (SPE)-based sample preparation protocols (comprising SOLAµTM HRP SPE spin plates from Thermo Fisher Scientific and ZIPTIP® C18 pipette tips from Merck Millipore) for analytical performance, reproducibility and analysis speed. The house swine represents a promising animal model for studying human eye diseases including glaucoma and provides excellent requirements for the qualitative and quantitative MS-based comparison in terms of ocular proteomics. In total 6 technical replicates of two protein fractions [extracted with 0.1% dodecyl-ß-maltoside (DDM) or 1% trifluoroacetic acid (TFA)] of porcine retinal tissues were subjected to in-gel trypsin digestion and purified with both SPE-based workflows (N=3) prior to LC-MS analysis. On average, 550±70 proteins (1512±199 peptides) and 305±48 proteins (806±144 peptides) were identified from DDM and TFA protein fractions, respectively, after ZIPTIP® purification, and SOLAµTM workflow resulted in the detection of 513±55 proteins (1347±180 peptides) and 300±33 proteins (722±87 peptides), respectively (FDR<1%). Venn diagram analysis revealed an average overlap of 65±2% (DDM fraction) and 69±4% (TFA fraction) in protein identifications between both SPE-based methods. Quantitative analysis of 25 glaucoma-related protein markers also showed no significant differences (P>0.05) regarding protein recovery between both SPE methods. However, only glaucoma-associated marker MECP2 showed a significant (P=0.02) higher abundance in ZIPTIP®-purified replicates in comparison to SOLAµTM-treated study samples. Nevertheless, this result was not confirmed in the verification experiment using in-gel trypsin digestion of recombinant MECP2 (P=0.24). In conclusion, both SPE-based purification methods worked equally well in terms of analytical performance and reproducibility, whereas the analysis speed and the semi automation of the SOLAµTM spin plates workflow is much more convenient in comparison to the ZIPTIP® C18 method.