MStern blotting-high throughput polyvinylidene fluoride (PVDF) membrane-based proteomic sample preparation for 96-well plates Journal Article
| Authors: | Berger, S. T.; Ahmed, S.; Muntel, J.; Polo, N. C.; Bachur, R.; Kentsis, A.; Steen, J.; Steen, H. |
| Article Title: | MStern blotting-high throughput polyvinylidene fluoride (PVDF) membrane-based proteomic sample preparation for 96-well plates |
| Abstract: | We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; human cell; protein localization; protein degradation; high throughput screening; proteomics; protein processing; uterine cervix cancer; western blotting; intermethod comparison; tandem mass spectrometry; urinalysis; protein urine level; liquid chromatography; molecular weight; physical chemistry; cell lysate; ovary cyst; hydrophobicity; protein cerebrospinal fluid level; ultrafiltration; artificial membrane; limit of quantitation; human; female; priority journal; article; polyvinylidene fluoride; squamous cell carcinoma antigen; filter assisted sample preparation; mstern blotting; protein blotting |
| Journal Title: | Molecular & Cellular Proteomics |
| Volume: | 14 |
| Issue: | 10 |
| ISSN: | 1535-9476 |
| Publisher: | Amer Soc Biochemistry Molecular Biology Inc |
| Date Published: | 2015-10-01 |
| Start Page: | 2814 |
| End Page: | 2823 |
| Language: | English |
| DOI: | 10.1074/mcp.O115.049650 |
| PROVIDER: | scopus |
| PMCID: | PMC4597154 |
| PUBMED: | 26223766 |
| DOI/URL: | |
| Notes: | Export Date: 2 November 2015 -- Source: Scopus |
