Accurate determination of protein phosphorylation is challenging, particularly 
for researchers who lack access to a high-accuracy mass spectrometer. In this 
study, multiple protocols were used to enrich phosphopeptides, and a rigorous 
filtering workflow was used to analyze the resulting samples. Phosphopeptides 
were enriched from cultured rat renal proximal tubule cells using three commonly 
used protocols and a dual method that combines separate immobilized metal 
affinity chromatography (IMAC) and titanium dioxide (TiO(2)) chromatography, 
termed dual IMAC (DIMAC). Phosphopeptides from all four enrichment strategies 
were analyzed by liquid chromatography-multiple levels of mass spectrometry 
(LC-MS(n)) neutral-loss scanning using a linear ion trap mass spectrometer. 
Initially, the resulting MS(2) and MS(3) spectra were analyzed using 
PeptideProphet and database search engine thresholds that produced a false 
discovery rate (FDR) of <1.5% when searched against a reverse database. However, 
only 40% of the potential phosphopeptides were confirmed by manual validation. 
The combined analyses yielded 110 confidently identified phosphopeptides. Using 
less-stringent initial filtering thresholds (FDR of 7-9%), followed by rigorous 
manual validation, 262 unique phosphopeptides, including 111 novel 
phosphorylation sites, were identified confidently. Thus, traditional methods of 
data filtering within widely accepted FDRs were inadequate for the analysis of 
low-resolution phosphopeptide spectra. However, the combination of a streamlined 
front-end enrichment strategy and rigorous manual spectral validation allowed 
for confident phosphopeptide identifications from a complex sample using a 
low-resolution ion trap mass spectrometer.