Ok Thank you. that makes sense. I should filter the list first before Stpeter quantitation.
On Thursday, August 24, 2023 at 3:05:19 PM UTC-7 [email protected] wrote: > Hi Sudar, > > I'm catching up here... > > It looks like you've set your analysis pipeline properly - it is the same > way I would do it. One recommendation, though, is to do the quantitation at > an appropriate FDR threshold (i.e. FDR=0.01) instead of on all proteins. > This is because SIn is normalized on all quantified signals. So including > all protein groups means you are normalizing your quantities on proteins > that are also not really in your sample. > > As for the reason that some of your samples have fewer proteins than > others, there are several possibilities, but they are unlikely to be > related to StPeter. It could be, for example, that some of the charge state > models failed in PeptideProphet, or that several high-probability decoy > sequences skewed the distributions. Or perhaps a few key nondegenerate > peptides were not observed, causing ProteinProphet to group proteins in > drastically different ways. Or there are simply fewer proteins in those > samples (number of spectra need not correlate to number of proteins). There > isn't any one possible explanation without actually exploring the data, as > each dataset will have its own characteristics that can influence the > analysis. > > Cheers, > Mike > > On Friday, August 18, 2023 at 11:36:41 AM UTC-7 sudarshan kumar wrote: > >> Hi Mike, thank you I did the way you said. Analyzed all samples >> separately. >> I have a question >> I kept on doing as >> from mzml convert --- comet search -- peptide prophet---- iprophet ---- >> protein prophet---- stpeter. >> I didn't check the data quality neither filtered my data for anything >> during the process. >> Can I do filtering in the end, based on protein probability as decided by >> the sensitivity and error table? I did filtering at the end choosing error >> rate of 0.05. >> I had 6 files of similar samples. I see that based on stpeter score the >> top hits (around 30 proteins) are similar across 6 samples. But the total >> number of proteins identified in each sample goes down drastically in few >> of the samples although all the files had similar number of displayed >> spectra upto iprophet. >> CAn you please explain th reason? >> thanks >> sud >> >> >> >> On Thu, Aug 17, 2023, 10:15 sudarshan kumar <[email protected]> wrote: >> >>> OK, I can understand. so if the SIn values (displayed) are log >>> transformed values. Can we take antilog of these values to come back to the >>> actual fractional values. >>> >>> Since, I want to compare two samples where i want to represent the >>> difference in the quantity of each protein as fold change. Am I needed to >>> do it separately in excel or other platform where i can calculate the >>> ration using fractional (normalized SIn values) for each of the proteins. >>> >>> Thank you >>> >>> On Wednesday, August 16, 2023 at 1:37:05 PM UTC-7 >>> [email protected] wrote: >>> >>>> Here's a detailed mathematical explanation of SIn: >>>> https://doi.org/10.1038/nbt.1592 >>>> >>>> Briefly, the normalization process produces fractional representation >>>> of quantities, i.e. 0.01, 0.052, etc. These are then log-transformed, >>>> which >>>> results in values that typically range from -5 to -30, but are always >>>> negative because they are log-transforms of values less than 1. >>>> >>>> These negative values can be adjusted by applying a scalar to the >>>> values (such as +30) so that all values are greater than 0. Alternatively, >>>> there is an option in StPeter (-s) where you can specify a total protein >>>> amount loaded into the instrument and StPeter will scale all the >>>> quantities >>>> to that amount. Note that this isn't an absolute value for each protein, >>>> because StPeter is standardizing the results to the sum of all *observed >>>> *proteins (not the ones you didn't identify), but it still puts all >>>> values in an easy to conceptualize, and positive, scale. >>>> >>>> These standardized numbers are comparable across samples (to obtain >>>> ratios) so long as the samples were acquired with the same instrument >>>> method and chromatography conditions. >>>> >>>> Cheers, >>>> Mike >>>> >>>> On Wednesday, August 16, 2023 at 10:53:34 AM UTC-7 sudarshan kumar >>>> wrote: >>>> >>>>> and >>>>> in StPeter what does negative SIn scor emean? I see all of them being >>>>> negative >>>>> >>>> -- >>> You received this message because you are subscribed to the Google >>> Groups "spctools-discuss" group. >>> To unsubscribe from this group and stop receiving emails from it, send >>> an email to [email protected]. >>> To view this discussion on the web visit >>> https://groups.google.com/d/msgid/spctools-discuss/af5ca34a-32b9-476e-98cd-185c21b6c483n%40googlegroups.com >>> >>> <https://groups.google.com/d/msgid/spctools-discuss/af5ca34a-32b9-476e-98cd-185c21b6c483n%40googlegroups.com?utm_medium=email&utm_source=footer> >>> . >>> >> -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To unsubscribe from this group and stop receiving emails from it, send an email to [email protected]. To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/9f9317da-2e02-4983-ae6c-0377c20f0d9dn%40googlegroups.com.
