Speed Through your LC-MS Sample Prep with New High Throughput Filtration Step
Modified filter-aided sample preparation (FASP) protocol uses Pall AcroPrep™ Advance Filter Plates to improve LC-MS results with a dramatic reduction in sample prep time
September 9, 2021
As we discussed in the first blog in this series, sample filtration has been shown to be a powerful tool for improving experimental results in liquid chromatography-mass spectrometry (LC-MS), provided you select the correct filter. In this second part, we look at sample filtration in a high-throughput setting using Pall’s AcroPrep 96-well plates, and a new protocol that shaves hours off the typical LC-MS workflow.
LC-MS is a powerful, data-rich assay platform with a broad range of applications. However, typical low volume LC-MS requires the manual or semi-automated addition of analytical samples onto the HPLC column, followed by up to 15 minutes of run time on the HPLC column before the sample is eluted into the mass spec via electrospray. In order to take advantage of LC-MS in high volume applications, a level of automation and standardization of the sample preparation process using 96-well plates is required. In this blog, we discuss a recently published, modified FASP protocol that takes advantage of the characteristics of the Pall AcroPrep 96-well filter plates to prepare high-quality samples for LC-MS in a high volume, high-throughput setting. This modified process reduces the total protocol time by up to three hours, without any loss of sample quality, and has since been adopted into a wide range of research fields and applications, as evidenced by recent publications(2, 3, 4).
High throughput 96-well sample prep
Filter-aided sample preparation (FASP) is a common technique used to prepare samples for HPLC and LC-MS. Potriquetet al. published a protocol in the Journal PLoS One detailing an accelerated method of plate-based FASP using AcroPrep Advance 96-well Omega™filter plates(1). This improved protocol allowed the researchers to shave three hours off their typical LC-MS sample prep workflow for high-throughput proteomic analyses, a significant improvement. As well as the time saving, the team was able to demonstrate comparable results to those obtained using more traditional longer duration protocols.
The key to the time savings was the switch to AcroPrep Advance 96-well Omega filter plates which utilize a modified polyethersulfone (PES) molecular weight cut-off membrane instead of the typical cellulose membrane used on this type of filter plate. The PES membrane enabled the team to use isopropanol as a wetting agent, which decreased spin times required for buffer exchange from an hour to 30 minutes. Over the course of a typical lab protocol, this adds up to a substantial three-hour time saving.
To test the performance of their accelerated FASP protocol, the researchers compared the results to the original (longer duration) FASP, as well as to other alternative methods including eFASP and MStern-blot. The results showed comparable performance across all five methods in terms of both reproducibility and the range of proteins detected by the HPLC, demonstrating that accelerated FASP using AcroPrep Advance 96-well Omega filter plates is a real alternative to traditional, more time-consuming methods of LC-MS sample prep.
The modified FASP method using Pall AcroPrep plates has been incorporated into a range of different scientific methods and protocols. In 2021, researchers used AcroPrep Advance 96-well Omega filter plates in a modified FASP protocol as part of their sample prep for LC-MS/MS in a study to identify new drug candidates against melanoma(2). In addition, scientists incorporated AcroPrep plates into a sample prep protocol for high-throughput urine proteomics(3), and another group used the AcroPrep plates in a protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS(4).
Interested in accelerating your LC-MS sample prep? Check out the AcroPrep Advance 96-well Omega Filter Plates
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- Potriquet J, Laohaviroj M, Bethony JM, Mulvenna J (2017) A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry. PLoS ONE 12(7): e0175967. https://doi.org/10.1371/journal.pone.0175967
- Javier Moral-Sanz, Manuel A. Fernandez-Rojo, Jeremy Potriquet, Pamela Mukhopadhyay, Andreas Brust, Patrick Wilhelm, Taylor B. Smallwood, Richard J. Clark, Bryan G. Fry, Paul F. Alewood, Nicola Waddell, John J. Miles, Jason P. Mulvenna, and Maria P. Ikonomopoulou. ERK and mTORC1 Inhibitors Enhance the Anti-Cancer Capacity of the Octpep-1 Venom-Derived Peptide in Melanoma BRAF(V600E) Mutations. Toxins (Basel). 2021 Feb; 13(2): 146. Published online 2021 Feb 14. doi: 10.3390/toxins13020146
- Ahmed S., Fatou B., Mehta N.M., Bennike T.B., Steen H. (2021) Sample Preparation for High-Throughput Urine Proteomics Using 96-Well Polyvinylidene Fluoride (PVDF) Membranes..In: Baptista Carreira dos Santos H.M. (eds) Translational Urinomics. Advances in Experimental Medicine and Biology, vol 1306, May 2021. Springer, Cham. https://doi.org/10.1007/978-3-030-63908-2_1
- SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS. Kamil Mikulášek, Hana Konečná, David Potěšil, Renata Holánková, Jan Havliš and ZbyněkZdráhal. Front. Plant Sci., 10 March 2021 | https://doi.org/10.3389/fpls.2021.635550