Vignettes
This page will walk you through using the FlashLFQ graphical user interface (GUI) with a standard set of files we have provided.
First, click this link to view the latest release of FlashLFQ. Download FlashLFQ by clicking "FlashLFQ.zip" and extract the files to a folder. You do not need to install anything; just open FlashLFQ by starting GUI.exe.
Then, download the vignette files: https://uwmadison.box.com/s/w9bifgj7q8kwn9019r3b8u2y19n7hd2h
There are two spectra files in Thermo .raw format: 09-04-18_EcoliSpikeInSingleShot1x.raw and 09-04-18_EcoliSpikeInSingleShot2x.raw. The first file is a sample consisting of a mixture of E. coli and human (K562 cell line) lysate, digested with trypsin. The second sample is similar, except that the amount of E. coli lysate is double that of the first sample. If FlashLFQ does its job, then you should see E. coli peptides/proteins doubling in abundance, with human proteins staying constant (with a bit of noise).
We will provide FlashLFQ with a list of peptides identified in the samples. FlashLFQ will quantify those peptides, then quantify the proteins associated with those peptides. The peptides were identified with MetaMorpheus.
In the FlashLFQ folder, start GUI.exe. You should see a screen like this:
On the left hand side, navigate to "Identifications". Find the AllPSMs.psmtsv file in the FlashLFQVignette\MetaMorpheus Search Results\Task1-SearchTask folder. You can either drag-and-drop this file into the FlashLFQ program or click the "Add Identifications" button to browse to it. Once you've added the identification file, your screen should now look like this:
Navigate to the Spectra tab on the left. Add the two .raw spectra files into FlashLFQ by drag-and-drop or browsing via the "Add Spectra" button. A window asking you to accept Thermo's licence to use its RawFileReader software may appear. RawFileReader is used by FlashLFQ to read Thermo .raw files in their native form. Accept the licence to continue.
Your screen should look like this:
You will now need to specify the experimental design. This is needed to perform the normalization and statistical analysis steps in FlashLFQ. It also helps match-between-runs. If you do not want use these features of FlashLFQ, you can skip the experimental design step.
The cells under "Condition", "Sample", "Fraction", and "Replicate" are editable. FlashLFQ's statistical analysis determines the change in protein abundance between conditions. In this analysis, we want to determine the change in protein abundance between the "Ecoli 1x" sample and the "Ecoli 2x" sample. Thus, we will define these two conditions, with one sample per condition. Change the SingleShot1x file's condition to something like "Ecoli 1x" and the SingleShot2x file's condition to something like "Ecoli 2x". Change both sample numbers to "1" (meaning that they are sample #1 of their respective conditions).
Your screen should look like this:
Navigate to "Settings" on the left. This is the area that specifies FlashLFQ's run settings. In this analysis, we will perform intensity normalization, match-between-runs, and the Bayesian statistical analysis. Check these boxes. The "Control Condition" is the condition to quantify the change in protein abundance relative to; in this case, we want to know which proteins are changing in the 2x sample relative to the 1x sample, so we will select the "Ecoli 1x" condition as the control. The fold-change cutoff in log2 units is what is considered to be "noise" (biological and technical). You can specify this manually or have FlashLFQ determine a cutoff for you. I have chosen a manual cutoff of 0.2 here.
Your screen should look like this:
Finally, navigate to the "Run" tab on the left. An output directory should be specified, but if you would like to change it you may do so. Then click the "Run FlashLFQ" button.
After a couple of minutes, FlashLFQ should finish.
In the output folder, you should have a list of quantified chromatographic peaks, peptides, and proteins (along with statistical significance for the protein fold-changes).
The output folder contains a list of FlashLFQ's settings in a file called FlashLfqSettings.toml It is a plain text file that can be edited in, for example, Notepad. At some point, you may wish to reproduce someone else's quantitative analysis (or someone may wish to reproduce yours). This settings file provides a means to do so.
There are many ways to analyze FlashLFQ's quantitative output. A few examples are provided here. Plots were generated using OriginLab.
Volcano plot:
Fold-change boxplots (only proteins with >1 fold-change measurement were plotted):
Sensitivity vs. Specificity curve (only proteins with >1 fold-change measurement were plotted):
