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vdemichev / DiaNN

DIA-NN - a universal automated software suite for DIA proteomics data analysis.
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MaxLFQ parameters #471

Open Cobboldss opened 2 years ago

Cobboldss commented 2 years ago

Hi. I was interested in understanding in more detail how DIA-NN implements the MaxLFQ algorithm and if it is possible to specify certain parameters in the command box.

I understand 5 fragment ions are used for peptide quantification but what are the default parameters for protein quantification (i.e. minimum peptide amount, use only unique peptides)?

In a separate 'issue' discussion it was described that a Top(N) command can be made, can something similar be done with MaxLFQ to specify the minimum number of peptides to use for protein quantification?

Thanks in advance,

Simon

vdemichev commented 2 years ago

Hi Simon,

Top 3 fragment ions are used for the peptide by default. All peptides match to the protein group in the main report are used for quantification of proteins. All peptides uniquely matched to the given gene are used to calculate Genes.MaxLFQ.Unique. You can choiose peptides yourself and do MaxLFQ in RT also using iq or diann R packages.

MaxLFQ works very well without extra prefiltering of peptides, so there's no such built-in possibility, but can always to it in R of course.

Best, Vadim

Cobboldss commented 2 years ago

Hi Vadim,

Thanks for clarifying. That is very helpful.

Best,

Simon

diabeginner01 commented 2 years ago

Hi Simon,

Top 3 fragment ions are used for the peptide by default. All peptides match to the protein group in the main report are used for quantification of proteins. All peptides uniquely matched to the given gene are used to calculate Genes.MaxLFQ.Unique. You can choiose peptides yourself and do MaxLFQ in RT also using iq or diann R packages.

MaxLFQ works very well without extra prefiltering of peptides, so there's no such built-in possibility, but can always to it in R of course.

Best, Vadim

Hi Vadim, regarding this reply above I have some questions. I understood that top 6 fragments are used, as the instructional materials state in the list of commands: "--quant-fr [N] sets the number of top fragment ions among which the fragments that will be used for quantification are chosen. Default value is 6" So is it 6 or 3? And is it top N fragments per precursor or peptide (which might have multiple different charge state precursors)?

Also: "All peptides match to the protein group in the main report are used for quantification of proteins." Can I also instruct DIA-NN to use only top 3 peptides with the highest fragment intensities for quantification instead of all peptides?

Thanks for any clarification!

vdemichev commented 2 years ago

Top 3 fragments, based on average correlation scores across all runs, are chosen among the top 6 fragments from the library, wherein fragments are ordered by their reference intensities. All quant is precursor-level. No, it's beneficial to use all peptides.

GianArauz commented 1 year ago

Hi @vdemichev,

Just another quick question in the same line than @Cobboldss's question and your original answer:

All peptides match to the protein group in the main report are used for quantification of proteins.

This means that non-unique peptides are taken into account when using MaxLFQ algorithm for protein quantification?

Many thanks in advance for your time and for developing/sharing DIA-NN!

Gian.