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Extending the identification coverage of hydrophilic proteome and phosphoproteome via a graphite solid phase extraction StageTip

ABSTRACT: Desalting and concentrating peptides using reverse phase chromatography prior LC-MS/MS analysis is a critical step to improve the sensitivity of peptide detection. However, hydrophilic peptides are not easy to retain on the conventional solid phase extraction (SPE) StageTip using C18 as materials during the desalting step. In this study, graphite carbon was utilized as a SPE StageTip with strong hydrophobic adsorbent, which doubled the identification number of multiply phosphopeptides compared to the number using C18 SPE StageTip. In addition, (phospho)peptides with lower GRAVY value and shorter peptides length were enriched using the graphite SPE StageTip. Finally, a total of 7,617 phosphopeptides were identified, which covers both hydrophilic and hydrophobic properties. The complementary affinity of different desalting materials including C18, SDB-XC, and graphite was further demonstrated to deepen the coverage of proteome and phosphoproteome.

ORGANISM(S): Homo Sapiens (human) Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Andy W. Tao

PROVIDER: PXD018699 | JPOST Repository | Wed Apr 21 00:00:00 BST 2021

REPOSITORIES: jPOST

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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.

			Action	DRS
	031118-IMAC-C18.raw	Raw
	031118-IMAC-Graphite.raw	Raw
	031118-IMAC-SDB.raw	Raw
	032118-C18-FT-10ACN.raw	Raw
	032118-C18-FT-15ACN.raw	Raw

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Extending the identification coverage of hydrophilic proteome and phosphoproteome via a graphite solid phase extraction StageTip

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