As a follow up to my last blog post on analysis of fractionated samples using Progenesis LC-MS, here is a published example. Geun-Cheol Gil, Jim Brennan, Dan Throckmorton, Steven Branda and Gabriela Chirica from the Systems Biology department at Sandia National Laboratories (CA, USA) studied the mouse serum proteome using a fractionation approach1.
The study used a customised chromatography system for reproducible fractionation. The fractions were analysed by nanoLC-MS with data analysis using the fractionation workflow in Progenesis LC-MS. I spoke with one of the authors, Dr Geun-Cheol Gil, who said:
“We successfully analysed low molecular weight, low abundant peptides in a complex sample using a reliable fractionation process coupled with analysis by label-free nanoLC-MS. Progenesis LC-MS enabled us to analyse the label-free data from fractionated samples in a rigorous and reliable way and help us to generate a global view of the “peptidome” within mouse serum. Our platform, including this software, and our approach also show that by running simple checks you can minimise the fractions needed to achieve good peptidome coverage and so save instrument time and costs in achieving experimental goals.”
The mouse serum includes components with a wide range of MW and concentration of components. The group wanted to specifically investigate the peptidome (low MW, low abundant peptides in the body fluid). They were able to achieve a comprehensive peptidome identification of 357 proteins, covering a range of biological functions.
3-D montages of the peptide QSENVGLSSELNR of Testis-specific serine/threonine-protein kinase (accession # TSSK1_Mouse) on Progenesis LC-MS. The peptide was generated by trypsin digestion and ANOVA test was performed for the three sample sets. The p value was 0.06 for the peptide1. Each peak within the green outline represents a single isotope of the same peptide ion.
While fractionation offers a route to increase protein and proteome coverage, especially of low abundance components, it can rapidly increase the number of runs needed per experiment. But due to cost and time demands on analysis, it is desirable to minimise the number of fractions used. In the published study the final results were based on 3 experiments x 4 fractions x 3 repeats, so a total of 36 LC-MS/MS runs.
This number of fractions and repeats was not arbitrary. The study included analysis to determine how many repeats were needed to provide reliable coverage as well as checks on how well the fractionation had worked. A run of 6 replicates was made and coverage achieved in the first replicate (89%) was compared to coverage in subsequent replicates. It was a step worth taking, since no further increase in proteome coverage was seen beyond 3 replicates. So, the group could be confident in achieving maximum coverage with a relatively low number of replicates.
You can also check to make sure how well your fractionation procedure performs. This was achieved by comparing the distribution of identified proteins between each fraction. This was illustrated in the study by the figure below. Ideally the overlap should be minimal.
Distribution of identified proteins among RAM-RP fractions F1, F2, F3 and F4, generated by elution buffers containing 30, 40, 60 and 80% methanol in 0.1% formic acid, respectively 1.
The final Review Proteins screen can also display this information directly in Progenesis LC-MS. The table of results illustrates, with the numbered blue squares, which fractions each protein identification is based on.
An example not from the publication.
You can also use the peptide fraction chart to show if you have achieved an even coverage of peptides across all fractions or discover if the same quality of results could be delivered with fewer fractions. In the example below then you could remove at least the first fraction and perhaps optimise the fractionation step to generate equal numbers of peptides or proteins per fraction?
An example not from the publication.
In the words of the authors in this published study:
“Platforms which enable comprehensive, high-throughput and time course analysis of bodily fluids in a cost-effective manner can establish proteomic mass spectrometric survey as the method of choice for personalised diagnostics, monitoring and treatment.”
It is reassuring to see the fractionation workflow in Progenesis LC-MS was considered a key component in this platform and the study provides the first published example of this application. Download Progenesis LC-MS and try analysing some of your fractionated samples for FREE.
1. G.-C. Gil, J. Brennan, D.J. Throckmorton, S.S. Branda, G.S. Chirica, Automated analysis of mouse serum peptidome using restricted access media and nanoliquid chromatography-tandem mass spectrometry, Journal of Chromatography B (2010), doi:10.1016/j.jchromb.2011.03.028