same peptide seq but different STY localization probability of same Serine amino acid

[singhsk2622]

Hi, I did phospho analysis in Fragpipe-DIA-NN.
I see one strange thing in output file. I see that for same protein there 3 STY peptides has been identified. All three peptides have same sequence only difference in localization probability of same Serine amino acid. why it is assigning three different peptides and assigning in different rows? Any one explain it me?

[vdemichev]

Hi,

Can you please paste here the whole rows you are showing along with column names?

Best,
Vadim

[singhsk2622]

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Protein.Group	Protein.Ids	Protein.Names	Genes	First.Protein.Description	Proteotypic	Stripped.Sequence	Modified.Sequence	Precursor.Charge	Precursor.Id	E:\DATA\Mette_sty_4343-4375\H2_ANP_Slot2-33_1_4351.d	E:\DATA\Mette_sty_4343-4375\H1_CTRL_Slot2-28_1_4341.d	E:\DATA\Mette_sty_4343-4375\H5_CNP_Slot2-41_1_4367.d	E:\DATA\Mette_sty_4343-4375\H6_CNP_Slot2-44_1_4373.d	E:\DATA\Mette_sty_4343-4375\H1_CNP_Slot2-29_1_4343.d	E:\DATA\Mette_sty_4343-4375\H6_ANP_Slot2-45_1_4375.d	E:\DATA\Mette_sty_4343-4375\H2_CNP_Slot2-32_1_4349.d	E:\DATA\Mette_sty_4343-4375\H5_ANP_Slot2-42_1_4369.d	E:\DATA\Mette_sty_4343-4375\H11_ANP_Slot2-30_1_4345.d	E:\DATA\Mette_sty_4343-4375\H2_CTRL_Slot2-31_1_4347.d	E:\DATA\Mette_sty_4343-4375\H4_ANP_Slot2-39_1_4363.d	E:\DATA\Mette_sty_4343-4375\H5_CTRL_Slot2-40_1_4365.d	E:\DATA\Mette_sty_4343-4375\H3_CTRL_Slot2-34_1_4353.d	E:\DATA\Mette_sty_4343-4375\H3_ANP_Slot2-36_1_4357.d	E:\DATA\Mette_sty_4343-4375\H6_CTRL_Slot2-43_1_4371.d	E:\DATA\Mette_sty_4343-4375\H4_CNP_Slot2-38_1_4361.d	E:\DATA\Mette_sty_4343-4375\H4_CTRL_Slot2-37_1_4359.d	E:\DATA\Mette_sty_4343-4375\H3_CNP_Slot2-35_1_4355.d	All Mapped Proteins	All Mapped Genes	M:15.9949	M:15.9949 Best Localization	M:15.9949 Best Scan	STY:79.96633	STY:79.96633 Best Localization	STY:79.96633 Best Scan	n:42.0106	n:42.0106 Best Localization	n:42.0106 Best Scan
F1M6Z4	F1M6Z4		Prob1		1	AASAPPTDRLHPASSLGMR	AAS(UniMod:21)APPTDRLHPASSLGM(UniMod:35)R	2	AAS(UniMod:21)APPTDRLHPASSLGM(UniMod:35)R2	4743.61	1802.44	620.495	1679.9	3714.83	2351.35	2216.74	1634.64	4171.05	2327.22	1572.71	593.243	616.555	2498.75		2767.73	1533.18		tr|F1M6Z4|F1M6Z4_RAT	Prob1	AASAPPTDRLHPASSLGM(1.0000)R	1	H1_CNP_Slot2-29_1_4343_diatracer.815118.815118.2	AAS(0.8971)APPT(0.0277)DRLHPAS(0.0379)S(0.0373)LGMR	0.8971	H1_CNP_Slot2-29_1_4343_diatracer.815118.815118.2
F1M6Z4	F1M6Z4		Prob1		1	AASAPPTDRLHPASSLGMR	AAS(UniMod:21)APPTDRLHPASSLGM(UniMod:35)R	3	AAS(UniMod:21)APPTDRLHPASSLGM(UniMod:35)R3	172206	54009.6	53949.4	99303.2	148441	120861	118466	78399.5	165968	118161	93134.4	72153.6	52809.7	103080	81127.9	127826	70269.4	64540.8	tr|F1M6Z4|F1M6Z4_RAT	Prob1	AASAPPTDRLHPASSLGM(1.0000)R	1	H11_ANP_Slot2-30_1_4345_diatracer.1040581.1040581.3	AAS(0.9302)APPT(0.0222)DRLHPAS(0.0239)S(0.0238)LGMR	0.9302	H4_CTRL_Slot2-37_1_4359_diatracer.782931.782931.3
F1M6Z4	F1M6Z4		Prob1		1	AASAPPTDRLHPASSLGMR	AAS(UniMod:21)APPTDRLHPASSLGM(UniMod:35)R	4	AAS(UniMod:21)APPTDRLHPASSLGM(UniMod:35)R4	3602.33			8367.68	3951.48	6857.46		5254.43	3482.65		3302.2								tr|F1M6Z4|F1M6Z4_RAT	Prob1	AASAPPTDRLHPASSLGM(1.0000)R	1	H5_ANP_Slot2-42_1_4369_diatracer.447942.447942.4	AAS(0.7913)APPT(0.0971)DRLHPAS(0.0391)S(0.0725)LGMR	0.7913	H5_ANP_Slot2-42_1_4369_diatracer.447942.447942.4

[vdemichev]

Charge states are different

[singhsk2622]

Should not be merged in final file? now when i am doing statistical analysis so i will have 3 different fold change for three same peptides with different charge. Any suggestion for this ?

[vdemichev]

You can either aggregate yourself based on the main report or (if using DIA-NN 1.9 or later) use the phosphosite_90/99 matrices, how these are calculated is described here https://github.com/vdemichev/DiaNN?tab=readme-ov-file#ptms-and-peptidoforms.

[singhsk2622]

thanks