ArticleAnalytical chemistry2012
Coupling methanol denaturation, immobilized trypsin digestion, and accurate mass and time tagging for liquid-chromatography-based shotgun proteomics of low nanogram amounts of RAW 264.7 cell lysate.
Article in Analytical chemistry, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 24 citations in OpenAlex.
- An automated spray-capillary platform for the microsampling and CE-MS analysis of picoliter- and nanoliter-volume samples.Analytical and bioanalytical chemistry · 2023Article
- Separation methods in single-cell proteomics: RPLC or CE?International journal of mass spectrometry · 2022Article
- Method for Identifying Galectin Ligands on Lymphocyte Membrane Glycoproteins.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Adductomics Pipeline for Untargeted Analysis of Modifications to Cys34 of Human Serum Albumin.Analytical chemistry · 2016Article
- Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.Analytical chemistry · 2016Article
- Uncovering immobilized trypsin digestion features from large-scale proteome data generated by high-resolution mass spectrometry.Journal of chromatography. A · 2014Article
- Insulator-based dielectrophoresis of mitochondria.Biomicrofluidics · 2014Article
- Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts.PloS one · 2014Article
- High sensitivity capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for the rapid analysis of complex proteomes.Current opinion in chemical biology · 2013Review
- Capillary zone electrophoresis-multiple reaction monitoring from 100 pg of RAW 264.7 cell lysate digest.The Analyst · 2013Article
- Integrated capillary zone electrophoresis-electrospray ionization tandem mass spectrometry system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of RAW 264.7 cell lysate.Analytical chemistry · 2013Article
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
We report the shotgun proteomic analysis of mammalian cell lysates that contain low nanogram amounts of protein. Proteins were denatured using methanol, digested using immobilized trypsin, and analyzed by UPLC-ESI-MS/MS. The approach generated more peptides and higher sequence coverage for a mixture of three standard proteins than the use of free trypsin solution digestion of heat- or urea-denatured proteins. We prepared triplicate RAW 264.7 cell lysates that contained 6, 30, 120, and 300 ng of protein. An average of 2 ± 1, 23 ± 2, 134 ± 11, and 218 ± 26 proteins were detected for each sample size, respectively. The numbers of both protein and peptide IDs scaled linearly with the amount of sample taken for analysis. Our approach also outperformed traditional methods (free trypsin digestion of heat- or urea-denatured proteins) for 6-300 ng RAW 264.7 cell protein analysis in terms of number of peptides and proteins identified. The use of accurate mass and time (AMT) tags resulted in the identification of an additional 16 proteins based on 20 peptides from the 6 ng cell lysate prepared with our approach. When AMT analysis was performed for the 6 ng cell lysate prepared with traditional methods, no reasonable peptide signal could be obtained. In all cases, roughly ∼30% of the digested sample was taken for analysis, corresponding to the analysis of a 2 ng aliquot of homogenate from the 6 ng cell lysate.
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