1.2 Prepare Reporter Ion Intensities
1.2.1 Read MASIC Output
MASIC is a tool for extracting ion intensities. With proper parameter settings, it can be used for extracting TMT (or iTRAQ) reporter ion intensities. In addition, it reports a number of other helpful metrics. Notably, the interference score at the precursor ion level and the signal-to-noise ratio (S/N) at the reporter ion level (computed by Thermo software). The interference score reflects the proportion of the ion population that was isolated for fragmentation that is due to the targeted ion. In other words, 1 - InterferenceScore is due to co-isolated species that have similar elution time and precursor ion m/z. The first step in the preparation of the reporter ion intensity data is to read the MASIC results. By default, the interference score is not included, so we need to set that argument to TRUE in order to filter the results after.
Similar to the MS-GF+ results, we can read the MASIC results from a local folder with PlexedPiper::read_masic_data or from PNNL’s DMS with PNNL.DMS.utils::read_masic_data_from_DMS.
## Get MASIC results from local folder - not run
# Get file path
path_to_MASIC_results <- "path_to_folder_containing_necessary_files"
# Read MASIC results from path
masic_data <- read_masic_data(path_to_MASIC_results,
interference_score = TRUE)## Get MASIC results from DMS
masic_data <- read_masic_data_from_DMS(data_package_num,
interference_score = TRUE)Normally, this would display progress bars in the console as the data is being fetched. However, the output was suppressed to save space.
Table 1.2 shows the first 6 rows of the unfiltered masic_data.
| Dataset | ScanNumber | Ion_126.128 | Ion_127.125 | Ion_127.131 | Ion_128.128 | Ion_128.134 | Ion_129.131 | Ion_129.138 | Ion_130.135 | Ion_130.141 | Ion_131.138 | Ion_126.128_SignalToNoise | Ion_127.125_SignalToNoise | Ion_127.131_SignalToNoise | Ion_128.128_SignalToNoise | Ion_128.134_SignalToNoise | Ion_129.131_SignalToNoise | Ion_129.138_SignalToNoise | Ion_130.135_SignalToNoise | Ion_130.141_SignalToNoise | Ion_131.138_SignalToNoise | InterferenceScore |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 2 | 70562.39 | 24864.62 | 17165.80 | 35625.00 | 92236.87 | 9640.23 | 8578.05 | 6996.69 | 11833.07 | 32281.34 | 71.47 | 25.17 | 17.38 | 36.04 | 93.32 | 9.75 | 8.67 | 7.07 | 11.96 | 32.71 | 0.996 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 3 | 23706.89 | 13559.32 | 5856.83 | 16322.71 | 34294.90 | 4853.11 | 7938.24 | 0.00 | 1465.03 | 18182.27 | 26.12 | 14.94 | 6.45 | 17.97 | 37.77 | 5.34 | 8.74 | NA | 1.61 | 19.93 | 0.993 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 4 | 12459.86 | 11785.91 | 10932.51 | 10653.32 | 12328.62 | 5959.86 | 9905.82 | 8387.04 | 11166.70 | 14053.40 | 12.40 | 11.75 | 10.90 | 10.64 | 12.31 | 5.96 | 9.91 | 8.40 | 11.18 | 14.13 | 1.000 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 5 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | 1.000 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 6 | 0.00 | 10998.67 | 0.00 | 21077.05 | 2725.50 | 0.00 | 0.00 | 0.00 | 0.00 | 6800.70 | NA | 9.19 | NA | 17.57 | 2.27 | NA | NA | NA | NA | 5.66 | 1.000 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 8 | 6166.82 | 1371.27 | 2418.35 | 8087.76 | 5485.35 | 0.00 | 0.00 | 1543.48 | 1943.96 | 7436.60 | 6.92 | 1.54 | 2.71 | 9.04 | 6.13 | NA | NA | 1.72 | 2.16 | 8.26 | 0.969 |
1.2.2 Filter MASIC Data
The only other step in reporter ion intensity data preparation is to filter the results. Currently, we recommend keeping entries where at least 50% of the ion population is due to the targeted ion (interference score \(\geq\) 0.5) and not filtering by S/N. To only reformat the data and not filter it, set both thresholds to 0.
# Filter MASIC data
masic_data <- filter_masic_data(masic_data,
interference_score_threshold = 0.5,
s2n_threshold = 0)Table 1.2 shows the first 6 rows of the filtered masic_data.
| Dataset | ScanNumber | Ion_126.128 | Ion_127.125 | Ion_127.131 | Ion_128.128 | Ion_128.134 | Ion_129.131 | Ion_129.138 | Ion_130.135 | Ion_130.141 | Ion_131.138 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 2 | 70562.39 | 24864.62 | 17165.80 | 35625.00 | 92236.87 | 9640.23 | 8578.05 | 6996.69 | 11833.07 | 32281.34 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 3 | 23706.89 | 13559.32 | 5856.83 | 16322.71 | 34294.90 | 4853.11 | 7938.24 | 0.00 | 1465.03 | 18182.27 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 4 | 12459.86 | 11785.91 | 10932.51 | 10653.32 | 12328.62 | 5959.86 | 9905.82 | 8387.04 | 11166.70 | 14053.40 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 5 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 6 | 0.00 | 10998.67 | 0.00 | 21077.05 | 2725.50 | 0.00 | 0.00 | 0.00 | 0.00 | 6800.70 |
| MoTrPAC_Pilot_TMT_W_S1_01_12Oct17_Elm_AQ-17-09-02 | 8 | 6166.82 | 1371.27 | 2418.35 | 8087.76 | 5485.35 | 0.00 | 0.00 | 1543.48 | 1943.96 | 7436.60 |
Lastly, we will save the processed MSnID and MASIC data to an .RData file with compression. This is useful in case we want to create different cross-tabs with new study design tables later on.
# Save processed MSnID and MASIC data
save(msnid, masic_data, file = "data/3442_processed_msnid_and_masic.RData",
compress = TRUE)