As input, proteiNorm
expects tab-separated peptide (optional) and protein data (not on
logarithmic scale) as produced by software such as MaxQuant,9 (link) where each row represents a peptide or protein
and the column names of the measured intensities (samples) beginning
with “Reporter intensity corrected” followed by an integer
and an optional label (e.g., “Reporter intensity corrected
5 TMT2”) for TMT experiments. The column names for samples
in label-free experiments should begin with “Intensity”
followed by an integer (“Intensity 01”). Data from both
mass spectrometry quantitation methods, tandem mass tag (TMT) and
label-free mass spectrometry, are supported. The TMT reporter intensities
should be corrected using the error correction factors provided by
Thermo Fisher for the specific TMT lot that was used during the sample
preparation. These correction factors are imported into MaxQuant when
setting up the database search parameters. The MaxQuant “proteinGroups.txt”
output file will then contain column names for each TMT reporter ion
labeled as “Reporter intensity corrected” and “Reporter
intensity”. In the following examples, we are using the “Reporter
intensity corrected” columns from MaxQuant since these intensities
apply the TMT correction factors. The reporter ion isotopic distributions
(−2, −1, +1, +2) are primarily for carbon isotopes with
reporter ion interference for each mass tag. ProteiNorm automatically
detects these column names and determines if the data is TMT or label-free
data type. ProteiNorm also removes proteins flagged as common contaminants
and reverse sequences in the MaxQuant output using the column names
“Potential contaminant” and “Reverse”
provided in the proteinGroups.txt MaxQuant output file. If you prefer
to load in a different matrix of data, then the column labels for
the samples will need to be modified to match the naming structure
provided here. An example of the data input files is hosted on Github
for both TMT and label-free data sets.
Due to the detection
limits of mass spectrometry instruments, many measurements of peptides
or proteins result in intensity levels of zero. These values will
be considered as missing values (denoted as NA) and can be imputed
with precaution. A modified heatmap of missing values (Figure 2 C) from the DEP Bioconductor
package10 (link) helps to determine if data is
missing at random (MAR) or missing not at random (MNAR), and the MSnbase
vignette describes different imputation methods used for different
types of missing data.11 (link)
expects tab-separated peptide (optional) and protein data (not on
logarithmic scale) as produced by software such as MaxQuant,9 (link) where each row represents a peptide or protein
and the column names of the measured intensities (samples) beginning
with “Reporter intensity corrected” followed by an integer
and an optional label (e.g., “Reporter intensity corrected
5 TMT2”) for TMT experiments. The column names for samples
in label-free experiments should begin with “Intensity”
followed by an integer (“Intensity 01”). Data from both
mass spectrometry quantitation methods, tandem mass tag (TMT) and
label-free mass spectrometry, are supported. The TMT reporter intensities
should be corrected using the error correction factors provided by
Thermo Fisher for the specific TMT lot that was used during the sample
preparation. These correction factors are imported into MaxQuant when
setting up the database search parameters. The MaxQuant “proteinGroups.txt”
output file will then contain column names for each TMT reporter ion
labeled as “Reporter intensity corrected” and “Reporter
intensity”. In the following examples, we are using the “Reporter
intensity corrected” columns from MaxQuant since these intensities
apply the TMT correction factors. The reporter ion isotopic distributions
(−2, −1, +1, +2) are primarily for carbon isotopes with
reporter ion interference for each mass tag. ProteiNorm automatically
detects these column names and determines if the data is TMT or label-free
data type. ProteiNorm also removes proteins flagged as common contaminants
and reverse sequences in the MaxQuant output using the column names
“Potential contaminant” and “Reverse”
provided in the proteinGroups.txt MaxQuant output file. If you prefer
to load in a different matrix of data, then the column labels for
the samples will need to be modified to match the naming structure
provided here. An example of the data input files is hosted on Github
for both TMT and label-free data sets.
Due to the detection
limits of mass spectrometry instruments, many measurements of peptides
or proteins result in intensity levels of zero. These values will
be considered as missing values (denoted as NA) and can be imputed
with precaution. A modified heatmap of missing values (
package10 (link) helps to determine if data is
missing at random (MAR) or missing not at random (MNAR), and the MSnbase
vignette describes different imputation methods used for different
types of missing data.11 (link)
