Raw MS Data (mzML) --> MaxQuant/DIA-NN --> proteinGroups.txt
                                                 |
                                                 v
            +--------------------------------------------+
            |             proteomics-pipeline            |
            +--------------------------------------------+
            |  1. Data Import & Filtering                |
            |  2. Log2 + inspect RAW distributions       |
            |  3. Normalization (after the inspection)   |
            |  4. Per-Group Completeness Filter          |
            |  5. QC: PCA, Correlation                   |
            |  6. Differential Abundance (limma/MSstats) |
            |  7. Visualization & Export                 |
            +--------------------------------------------+
                                                 |
                                                 v
                  Differential Proteins + Volcano Plots

library(limma)
library(ggplot2)
library(pheatmap)
 
# === 1. DATA IMPORT ===
proteins <- read.delim('proteinGroups.txt', stringsAsFactors = FALSE)
cat('Loaded', nrow(proteins), 'protein groups\n')
 
# Filter contaminants, reverse, only-by-site
proteins <- proteins[proteins$Potential.contaminant != '+' &
                      proteins$Reverse != '+' &
                      proteins$Only.identified.by.site != '+', ]
cat('After filtering:', nrow(proteins), 'proteins\n')
 
# Extract LFQ intensities
lfq_cols <- grep('^LFQ\\.intensity\\.', colnames(proteins), value = TRUE)
intensities <- proteins[, lfq_cols]
rownames(intensities) <- proteins$Majority.protein.IDs
colnames(intensities) <- gsub('LFQ\\.intensity\\.', '', colnames(intensities))
 
# === 2. LOG2 TRANSFORM, THEN INSPECT RAW DISTRIBUTIONS ===
intensities[intensities == 0] <- NA
log2_int <- log2(intensities)
 
# Inspect BEFORE normalizing (rule 4). Median-centering rescales every sample onto a common median,
# so it mathematically erases the 3x-low load that marks a failed injection -- after this point the
# failure is invisible. Identify and drop failures HERE.
boxplot(log2_int, las = 2, main = 'RAW log2 LFQ (pre-normalization)', ylab = 'log2 intensity')
id_counts <- colSums(!is.na(log2_int))
print(data.frame(id_count = id_counts, raw_median_log2 = round(apply(log2_int, 2, median, na.rm = TRUE), 2)))
 
# <50% of the cohort median ID count is a failed injection / low load, not biology.
failed <- names(id_counts)[id_counts < 0.5 * median(id_counts)]
if (length(failed) > 0) {
    message('Dropping failed samples: ', paste(failed, collapse = ', '))
    log2_int <- log2_int[, !colnames(log2_int) %in% failed, drop = FALSE]
}
 
# === 3. NORMALIZE (only after the raw inspection above) ===
sample_medians <- apply(log2_int, 2, median, na.rm = TRUE)
global_median <- median(sample_medians)
normalized <- sweep(log2_int, 2, sample_medians - global_median)
 
# === 4. FILTER ON PER-GROUP COMPLETENESS (do NOT impute by default) ===
# Filter FIRST on completeness PER GROUP: keep a protein if it is valid in >= ~50-70%
# of replicates in AT LEAST ONE condition. A protein missing in every group fails QC.
sample_info <- read.csv('sample_annotation.csv')
# Re-align the annotation to the samples that SURVIVED the raw-distribution QC above; otherwise the
# column indexing below requests a dropped sample and errors (or silently misaligns the design).
sample_info <- sample_info[sample_info$sample %in% colnames(normalized), ]
sample_info$condition <- droplevels(factor(sample_info$condition))
min_frac <- 0.6   # >= 60% present within at least one group; tune 0.5-0.7 per design
group_complete <- sapply(levels(sample_info$condition), function(g) {
    cols <- sample_info$sample[sample_info$condition == g]
    rowSums(!is.na(normalized[, cols, drop = FALSE])) >= ceiling(length(cols) * min_frac)
})
valid_rows <- rowSums(group_complete) > 0
filtered <- normalized[valid_rows, ]
cat('Proteins after per-group completeness filter:', nrow(filtered), '\n')
 
# Missingness in label-free DDA is left-censored MNAR (missing BECAUSE low). The modern,
# correct approach is to MODEL the missingness in the likelihood, NOT impute it. See
# proteomics/differential-abundance for the decision (proDA / msqrob2 / MSstats-AFT). The
# proDA path below is the RECOMMENDED route; the impute-then-limma path is a fallback.
 
# --- RECOMMENDED: model the missingness with proDA (no imputation) ---
# library(proDA)
# fit <- proDA(as.matrix(filtered), design = ~ condition, col_data = sample_info,
#              reference_level = 'Control')
# da <- test_diff(fit, contrast = 'conditionTreatment')   # columns: diff (log2FC), pval, adj_pval
# (Skip the === 5-6 impute/limma blocks below when using proDA.)
 
# --- FALLBACK ONLY: left-censored downshift imputation, then limma ---
# WARNING: downshift MANUFACTURES systematic false positives for on/off proteins near the
# detection limit (the volcano "anchor arms"): it pins missing values ~1.8 SD below the mean
# with an artificially tight 0.3 SD spread, inflating the t-statistic. The honest report for
# a protein fully missing in one group is "undetected in group B", NOT a fold change.
impute_minprob <- function(x) {
    nas <- is.na(x)
    if (all(nas)) return(x)
    x[nas] <- rnorm(sum(nas), mean = mean(x, na.rm = TRUE) - 1.8 * sd(x, na.rm = TRUE),
                    sd = 0.3 * sd(x, na.rm = TRUE))
    x
}
imputed <- as.data.frame(t(apply(filtered, 1, impute_minprob)))
 
# === 5. QC ===
# PCA
pca <- prcomp(t(imputed), scale. = TRUE)
pca_df <- data.frame(PC1 = pca$x[, 1], PC2 = pca$x[, 2], Sample = rownames(pca$x))
 
# === 6. DIFFERENTIAL ANALYSIS (fallback impute-then-limma path) ===
# sample_info is already loaded and factored in step 4. Put any batch in the design
# (~ batch + condition); removeBatchEffect() is visualization-only, never an input to lmFit.
design <- model.matrix(~ 0 + condition, data = sample_info)
colnames(design) <- levels(sample_info$condition)
 
fit <- lmFit(as.matrix(imputed), design)
contrast <- makeContrasts(Treatment - Control, levels = design)
fit2 <- contrasts.fit(fit, contrast)
 
# Select on FDR ALONE. A post-hoc fold-change + significance double filter inflates FDR
# (a collider/selection effect; realized FDR can exceed 50%). To require a minimum effect,
# use the moderated minimum-fold-change test treat()/topTreat() instead of filtering after.
fit2_treat <- treat(fit2, lfc = log2(1.5), trend = TRUE, robust = TRUE)   # moderated min-FC test; trend+robust ~mandatory for label-free LFQ
results <- topTreat(fit2_treat, coef = 1, number = Inf)
results$protein <- rownames(results)
results$significant <- results$adj.P.Val < 0.05
 
# === 7. OUTPUT ===
cat('\nResults:\n')
cat('  Significant proteins:', sum(results$significant), '\n')
cat('  Up-regulated:', sum(results$significant & results$logFC > 0), '\n')
cat('  Down-regulated:', sum(results$significant & results$logFC < 0), '\n')
 
write.csv(results, 'proteomics_results.csv', row.names = FALSE)

library(MSstats)
 
# From MaxQuant
evidence <- read.table('evidence.txt', sep = '\t', header = TRUE)
proteinGroups <- read.table('proteinGroups.txt', sep = '\t', header = TRUE)
annotation <- read.csv('annotation.csv')
 
# Convert to MSstats format
msstats_input <- MaxQtoMSstatsFormat(evidence = evidence,
                                      proteinGroups = proteinGroups,
                                      annotation = annotation)
 
# Process data
processed <- dataProcess(msstats_input, normalization = 'equalizeMedians',
                         summaryMethod = 'TMP', censoredInt = 'NA')
 
# Comparison. +1 on the numerator: Treatment=+1, Control=-1 so log2FC = Treatment - Control
# (positive = up in Treatment), matching the label and the limma makeContrasts(Treatment-Control) path.
comparison <- matrix(c(-1, 1), nrow = 1)
rownames(comparison) <- 'Treatment_vs_Control'
colnames(comparison) <- c('Control', 'Treatment')
 
results <- groupComparison(contrast.matrix = comparison, data = processed)

library(MSnbase)
 
# Extract reporter ions from spectra (NOT readMSnSet, which loads an existing text matrix)
raw <- readMSData('tmt.mzML', mode = 'onDisk')
tmt_data <- quantify(raw, reporters = TMT10, method = 'max')
# Correct isobaric impurity cross-talk with the LOT-SPECIFIC matrix from the reagent CoA.
# edit=FALSE avoids the interactive editor (default edit=TRUE blocks in scripts); load the CoA
# cross-talk values rather than the near-identity template makeImpuritiesMatrix(10) returns alone.
impurities <- makeImpuritiesMatrix(filename = 'tmt10_coa.csv', edit = FALSE)
tmt_data <- purityCorrect(tmt_data, impurities)
 
# Multi-batch TMT: do NOT concatenate plexes directly. Use MSstatsTMT, which applies the
# reference-channel (IRS) bridge during summarization:
#   library(MSstatsTMT)
#   summ <- proteinSummarization(msstatstmt_input)   # includes the cross-plex bridge
#   groupComparisonTMT(summ, contrast.matrix = comparison)

# SILAC ratios from MaxQuant
silac <- read.delim('proteinGroups.txt')
ratio_cols <- grep('Ratio.H.L.normalized', colnames(silac), value = TRUE)
 
# Log2 transform ratios
silac_log2 <- log2(silac[, ratio_cols])
 
# One-sample t-test against 0 (no change)
results <- apply(silac_log2, 1, function(x) t.test(x, mu = 0)$p.value)

library(arrow)
library(dplyr)
library(tidyr)
 
diann <- read_parquet('report.parquet')
 
# Filter to 1% FDR at precursor AND protein-group level before pivoting.
# Use the GLOBAL protein-group q-value for the cross-run matrix (per-run min(Q.Value) is anti-conservative).
# When MBR is ON, MBR has its own FDR: add the Lib.* q-values (Lib.Q.Value, Lib.PG.Q.Value <= 0.01).
diann_filt <- diann %>%
    filter(Q.Value <= 0.01 & PG.Q.Value <= 0.01 & Global.PG.Q.Value <= 0.01)
 
# PG.MaxLFQ is ALREADY cross-run MaxLFQ-normalized at report generation. Re-normalizing it
# double-normalizes -- go straight to log2 + limma with no further normalization. To apply the
# skill's own median-centering instead, pivot raw PG.Quantity here, not PG.MaxLFQ.
protein_matrix <- diann_filt %>%
    select(Protein.Group, Run, PG.MaxLFQ) %>%
    distinct() %>%
    pivot_wider(names_from = Run, values_from = PG.MaxLFQ)
 
# PG.MaxLFQ path: log2-transform and go straight to limma (no re-normalization)