Tyrosine phosphorylation plays a critical role in cell 
biological functions, particularly in signal transduction. 
Mass spectrometry (MS)-based proteomic analysis of 
tyrosine-phosphorylated (pTyr)
 proteins
 remains 
challenging due to their relatively low abundance. The 
methods of phosphopeptide enrichment are developed 
prosperously such as IMmuno Affinity Chromatography 
(IMAC) and Metal Oxide Affinity Chromatography 
(MOAC). However, the complexity of experimental design 
for sample separation induces the unnecessary variations, particularly in label-free study designs. Here, we report an 
alternative approach, Inclusion List-driven Targeted LC
tandem MS (ILT LC-tandem MS), and demonstrate the 
results
 obtained
 are comparable with that of 
phosphopeptide enrichment for increasing the sequencing 
possibility of low abundant pTyr peptides. In the present 
study, ILT LC-M/MS was applied for comparative label
free quantitative tyrosine phosphor proteomic analysis 
between Src inhibitor (dasatinib)-treated and untreated 
CL1-5 lung cancer cells. A total of 512 distinct pTyr sites 
corresponding to 491 peptides had been identified. According to evaluation using Mascot Delta score (MD
score), that 88.8% of the pTyr sites were considered 
confidently determined. In addition, the possibility of 
identifying multi phosphorylated peptides by ILT LC-
tandem MS was 11% higher than that by TiO2-based 
phosphopeptide enrichment previously reported. After the 
comparative label-free quantitative analysis, among the 
491 pTyr peptides identified 103 showed the significant 
level alteration of tyrosine phosphorylation between 
dasatinib-treated and untreated cells, and among which 53 
altered pTyr peptides were matched to contain Src kinase 
substrate motifs. Using pathway analysis of the 
corresponding proteins, the results illustrated that 17 
proteins were regulated by E2F transcription factor 1, 
which is known to be related to cell cycle regulation, and 
therefore provided a credible basis for further 
investigations. Our data demonstrate that the utility of an 
alternative approach, ILT LC--tandem MS, increases the 
sequencing possibility of low abundant pTyr peptides and 
reduces the induced variations in conventional TiO2 
enrichment label-free quantitative analysis.