Please keep only one intensity of listed features among multiple runs from one sample.

Tobias Kockmann

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Jan 3, 2017, 11:58:33 AM1/3/17

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Dear MStsats maintainers,

I am trying to process DIA data exported from Spectronaut 10, but I keep getting the following error message when calling:

> quant <- dataProcess(raw = pepitdeLevel.input)

** There are 1167 intensities which are zero or less than 1. These intensities are replaced with 1.

** Please check the listed featurues (A[+28]ALNLIR_2_b5noloss_1, A[+28]ALNLIR_2_y4noloss_1, A[+28]ALNLIR_2_y5noloss_1, A[+28]ALNLIR_2_y5noloss_2, A[+28]ALVDLEPGTMDSVR_2_y10noloss_1, A[+28]ALVDLEPGTMDSVR_2_y11noloss_1, A[+28]ALVDLEPGTMDSVR_2_y7noloss_1, A[+28]ALVDLEPGTMDSVR_2_y8noloss_1, A[+28]ALVDLEPGTMDSVR_2_y9noloss_1, A[+28]ANLIVEEEDLR_3_b5noloss_1, A[+28]ANLIVEEEDLR_3_b6noloss_1, A[+28]ANLIVEEEDLR_3_y4noloss_1, A[+28]ANLIVEEEDLR_3_y5noloss_1, A[+28]ANLIVEEEDLR_3_y6noloss_1, A[+28]ASIFGGAK[+28]PVDTAA

...

TLIC[+57]S_2_b4noloss_1, A[+28]VETLIC[+57]S_2_b5noloss_1, A[+28]VETLIC[+57]S_2_b6noloss_1, A[+28]VETLIC[+57]S_2_b6noloss_2, A[+28]VETLIC[+57]S_2_y7noloss_2, A[+28]VEVAPR_2_b3noloss_1, A[+28]VEVAPR_2_b7noloss_2, A[+28]VEVAPR_2_y4noloss_1, A[+28]VEVAPR_2_y5noloss_1, A[+28]VEVAPR_2_y6noloss_1, A[+28]VVASELR_2_b4noloss_1, A[+28]VVASELR_2_y4noloss_1, A[+28]VVASELR_2_y5noloss_1, A[+28]VVASELR_2_y6noloss_1, A[+28]VVASELR_2_y7noloss_1, A[+42]AAAAAError in dataProcess(raw = pepitdeLevel.input) :

Please keep only one intensity of listed features among multiple runs from one sample.

Can anyone explain we what the problem is? The input data looks like this:

> summary(pepitdeLevel.input)

ProteinName PeptideSequence

DK[+28]PLK[+28]DVIIVDSGK[+28]IEVEK[+28]PFAIAK[+28]E: 90 DK[+28]PLK[+28]DVIIVDSGK[+28]IEVEK[+28]PFAIAK[+28]E: 90

T[+28]PK[+28]FDQTFSAEWHQWK[+28]STHR : 90 T[+28]PK[+28]FDQTFSAEWHQWK[+28]STHR : 90

GENHC[+57]GIESEIVAGIPR : 78 GENHC[+57]GIESEIVAGIPR : 78

A[+28]TAEGPAAVTLAAPAFSR : 60 A[+28]TAEGPAAVTLAAPAFSR : 60

AAQQVSDSSSLLSQLR : 60 AAQQVSDSSSLLSQLR : 60

AAVPSGASTGIYEALELR : 60 AAVPSGASTGIYEALELR : 60

(Other) :92497 (Other) :92497

PrecursorCharge FragmentIon ProductCharge IsotopeLabelType Condition BioReplicate Run

2:50913 y6noloss:11079 1:68872 L:92935 C:41434 C1:14045 20150224_2_SWATH_C1:14045

3:29247 y7noloss: 9810 2:20759 G:51501 C2:14043 20150224_3_SWATH_G1:17242

4:10535 y5noloss: 9387 3: 3304 C3:13346 20150224_4_SWATH_C2:14043

5: 1636 y8noloss: 7573 G1:17242 20150224_6_SWATH_G2:17167

6: 574 y4noloss: 7053 G2:17167 20150224_8_SWATH_G3:17092

7: 30 y9noloss: 5951 G3:17092 20150224_9_SWATH_C3:13346

(Other) :42082

Intensity

Min. : 0.15

1st Qu.: 11.80

Median : 27.31

Mean : 94.77

3rd Qu.: 63.86

Max. :35623.43

I checked the listed features using:

> poi <- "A[+28]LYLVC[+57]GER"

> df <- droplevels(pepitdeLevel.input[pepitdeLevel.input$PeptideSequence == poi,])

> df <- unite(data = df, "featureID", 2:5, remove = FALSE)

> xtabs(~featureID+Run, df)

Run

featureID 20150224_2_SWATH_C1 20150224_3_SWATH_G1 20150224_4_SWATH_C2 20150224_6_SWATH_G2

A[+28]LYLVC[+57]GER_2_b3noloss_1 1 1 1 1

A[+28]LYLVC[+57]GER_2_y5noloss_1 1 1 1 1

A[+28]LYLVC[+57]GER_2_y6noloss_1 1 1 1 1

A[+28]LYLVC[+57]GER_2_y7noloss_1 1 1 1 1

A[+28]LYLVC[+57]GER_2_y8noloss_1 1 1 1 1

Run

featureID 20150224_8_SWATH_G3 20150224_9_SWATH_C3

A[+28]LYLVC[+57]GER_2_b3noloss_1 1 1

A[+28]LYLVC[+57]GER_2_y5noloss_1 1 1

A[+28]LYLVC[+57]GER_2_y6noloss_1 1 1

A[+28]LYLVC[+57]GER_2_y7noloss_1 1 1

A[+28]LYLVC[+57]GER_2_y8noloss_1 1 1

> xtabs(~featureID+BioReplicate, df)

BioReplicate

featureID C1 C2 C3 G1 G2 G3

A[+28]LYLVC[+57]GER_2_b3noloss_1 1 1 1 1 1 1

A[+28]LYLVC[+57]GER_2_y5noloss_1 1 1 1 1 1 1

A[+28]LYLVC[+57]GER_2_y6noloss_1 1 1 1 1 1 1

A[+28]LYLVC[+57]GER_2_y7noloss_1 1 1 1 1 1 1

A[+28]LYLVC[+57]GER_2_y8noloss_1 1 1 1 1 1 1

And I can not see any duplicated (non-unique entries). My data is should reflect a two-group comparision over 6 runs (3 runs per group), each replicate is covered by a single LC-MS run:

> xtabs(~Run+BioReplicate, pepitdeLevel.input)

BioReplicate

Run C1 C2 C3 G1 G2 G3

20150224_2_SWATH_C1 14045 0 0 0 0 0

20150224_3_SWATH_G1 0 0 0 17242 0 0

20150224_4_SWATH_C2 0 14043 0 0 0 0

20150224_6_SWATH_G2 0 0 0 0 17167 0

20150224_8_SWATH_G3 0 0 0 0 0 17092

20150224_9_SWATH_C3 0 0 13346 0 0 0

> xtabs(~Run+Condition, pepitdeLevel.input)

Condition

Run C G

20150224_2_SWATH_C1 14045 0

20150224_3_SWATH_G1 0 17242

20150224_4_SWATH_C2 14043 0

20150224_6_SWATH_G2 0 17167

20150224_8_SWATH_G3 0 17092

20150224_9_SWATH_C3 13346 0

Where is the problem? I have attached the complete log file.

Greetings,

Tobi

msstats-7.log

Tobias Kockmann

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Jan 16, 2017, 9:43:04 AM1/16/17

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Hello there,

It would be nice if somebody from the development team could at least give me a hint what the problem is about... I get the same error on a different machine and with an updated MSstats version (3.7.1).

Greetings,

Tobi

Meena Choi

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Jan 16, 2017, 11:53:11 PM1/16/17

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Dear Tobi,

It is difficult to know what the problem is, because I can't see how you assigned features.

I believe that SpectronauttoMSstatsformat function in v 3.7.3 can handle this issue. Please let us know if you still have the same error with the new version.

Sincerely,

- Meena

Chris Soon Heng Tan

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Feb 23, 2017, 9:02:36 PM2/23/17

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Dear Tobias

I am encountering the same problem asking me to "Please keep only one intensity of listed features among multiple runs from one sample". Did you manage to trace or solve the problem?

Many thanks

Chris

Meena Choi

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Feb 23, 2017, 9:13:30 PM2/23/17

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Dear Chris,

This type of issue can be happened in most of spectral processing tools except Skyline. From MSstats v3.7.3, it can be solved if you use the converter functions in MSstats for output from Skyline, Spectronaut, Progenesis, MaxQuant, Proteome Discoverer. (Please check MSstats manual from msstats.org) If you used other tool, please make sure that MSstats needs one row with one intensity value for one feature (PSM for DDA, transition level for SRM, fragmentation for DIA) and one MS run. This error message means there are more than one rows for one feature and one MS run.

If you have issue with converter functions, please share your data with us.

Hope to help.

Sincerely,

- Meena

Chris Soon Heng Tan

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Feb 24, 2017, 1:52:53 AM2/24/17

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Dear Meena

Thanks for your prompt response and advice. Will give it a try.

Chris

Edith Serrano

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Oct 26, 2017, 5:10:18 AM10/26/17

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Dear all,

I get the same message when using files processed through OpenSWATH and converted to MSstats input with SWATH2stats convert4MSstats function. Do you know if this converter tool is still compatible with MSstats?

Thanks,

Edith

Meena Choi

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Nov 22, 2017, 11:44:45 AM11/22/17

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My guess is that you may have fractionation. If yes, I'm not sure whether SWATH2stats works for fractionation case and I haven't had the chance to check the fractionation cases for SWATH2stats. Because MSstats needs more columns, 'Fraction' and 'techReplicate' in MSstats input for fractionation case.

If not fractionation, my guess that there is incorrect annotation for Run.

Hope to help.

- Meena

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