Error in Spectronaut input format

[sudip ghosh]

Hi Spectronaut team,

I am encountering error when importing from Spectronaut (v14).

it says:

INFO  [2022-01-18 20:45:57] ** Raw data from Spectronaut imported successfully.

Error in data.table::setnames(spec_input, c("PGProteinGroups", "EGModifiedSequence",  :
  'old' is length 8 but 'new' is length 9

for

spectronaut_imported_ELLU_proteome <- SpectronauttoMSstatsFormat(input,
+                            annotation = NULL,
+                            intensity = 'PeakArea',
+                            filter_with_Qvalue = TRUE,
+                            qvalue_cutoff = 0.01,
+                            useUniquePeptide = TRUE,
+                            fewMeasurements="remove",
+                            removeProtein_with1Feature = FALSE,
+                            summaryforMultipleRows=max)

> sessionInfo()
R version 4.1.1 (2021-08-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19042)

Matrix products: default

locale:
[1] LC_COLLATE=Swedish_Sweden.1252  LC_CTYPE=Swedish_Sweden.1252    LC_MONETARY=Swedish_Sweden.1252 LC_NUMERIC=C                  
[5] LC_TIME=Swedish_Sweden.1252    

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base    

other attached packages:
[1] MSstatsConvert_1.4.0 MSstats_4.1.1      

loaded via a namespace (and not attached):
 [1] Rcpp_1.0.8            pillar_1.6.4          compiler_4.1.1        nloptr_1.2.2.3        log4r_0.4.2           bitops_1.0-7        
 [7] tools_4.1.1           boot_1.3-28           lme4_1.1-27.1         checkmate_2.0.0       preprocessCore_1.56.0 lifecycle_1.0.1      
[13] tibble_3.1.6          nlme_3.1-155          gtable_0.3.0          lattice_0.20-45       pkgconfig_2.0.3       rlang_0.4.12        
[19] Matrix_1.4-0          ggrepel_0.9.1         vctrs_0.3.8           gtools_3.9.2          caTools_1.18.2        grid_4.1.1          
[25] glue_1.6.0            data.table_1.14.2     R6_2.5.1              marray_1.72.0         fansi_1.0.2           survival_3.2-13      
[31] limma_3.50.0          minqa_1.2.4           ggplot2_3.3.5         magrittr_2.0.1        backports_1.4.1       scales_1.1.1        
[37] gplots_3.1.1          ellipsis_0.3.2        MASS_7.3-55           splines_4.1.1         colorspace_2.0-2      utf8_1.2.2          
[43] KernSmooth_2.23-20    stringi_1.7.6         munsell_0.5.0         crayon_1.4.2 

the file headers in the input are:

 R.Condition.R.FileName.R.Replicate.PG.ProteinGroups.PG.Qvalue.PG.Quantity.PEP.GroupingKey.PEP.StrippedSequence.PEP.Quantity.EG.ModifiedSequence.EG.Qvalue.FG.Charge.F.Charge.F.FrgIon.F.NormalizedPeakArea.F.PeakArea

thanks,

Sudip

[Mateusz Staniak]

Hi,

what are the column names in your input data frame and what parameters do you use when calling SpectronauttoMSstatsFormat?

Kind regards,

Mateusz

[sudip ghosh]

Hi Mateusz,

I figured out that there was a mistake calling the input data. Its working now, but I found that the spectronaut default stat and MSStats group comparison output is matching. I guess there are different model fitting in the background, but still the group comparison output looks little strange! there is only one protein that passed the adjusted p value, whereas around 400 proteins passed the ordinary p value. when the checked the spectronaut output, there are many that passed the adjusted p! Could you please check the codes. this is a directDIA output between WT and mutant and both of them treated/untreaetd (4 conditions). I have attached the group comparison output. here are codes I used:

spectronaut_imported <- SpectronauttoMSstatsFormat(input,
                           annotation = NULL,
                           intensity = 'PeakArea',
                           filter_with_Qvalue = TRUE,
                           qvalue_cutoff = 0.01,
                           useUniquePeptide = TRUE,
                           fewMeasurements="remove",
                           removeProtein_with1Feature = FALSE,
                           summaryforMultipleRows=max,
                           use_log_file = FALSE)

spectronaut.proposed.normalized <- dataProcess(spectronaut_imported,
                                               normalization = "equalizeMedians",
                                               summaryMethod = "TMP",
                                               censoredInt = "0",
                                               MBimpute = FALSE,
                                               maxQuantileforCensored=0.999,
                                               use_log_file = FALSE)

comparison1<-matrix(c(-1,1,0,0),nrow=1)
comparison2<-matrix(c(0,0,-1,1),nrow=1)
comparison3<-rbind(comparison1,comparison2)

ELLU_proteome.comparisons1 <- groupComparison(contrast.matrix = comparison3,
                                             data = spectronaut.proposed.normalized,
                                             log_base = 2)

  

best,

Sudip