Lung cancer is the leading cause of cancer related deaths worldwide. Although treatment strategies have improved, overall survival has not significantly increased. Studies have revealed a heterogeneous subclonal architecture to lung cancer, which contributes to the persistence of the disease by allowing the tumour to metastasise to distant sites, utilising the different phenotypic characteristics of the heterogeneous population. The DLKP cell line was established from a lymph node metastasis of a primary lung tumour and is described as a poorly differentiated squamous lung carcinoma cell line. This cell line is comprised of three distinct subpopulations: DLKP-SQ, DLKP-M and DLKP-I, each with well-defined phenotypes. In vitro studies have shown DLKP-M to be extremely invasive relative to DLKP-SQ, whereas the latter cell line shows high anoikis resistance relative to DLKP-M. As the cells originate from the same tumour, these varied phenotypic characteristics make DLKP a particularly useful model for the study of cellular heterogeneity in lung cancer. Quantitative label-free LC-MS/MS was used to analyse the DLKP cell line and its subpopulations at a proteomic level. This resulted in a cohort of significantly differentially expressed proteins between the cell lines. From this, three differentially expressed proteins; Shootin-1 (highly expressed in DLKP-SQ), MARCKS (highly expressed in DLKP-M), and Desmoglein-3 (highly expressed in DLKP-I) were chosen for a series of follow up studies. The expression patterns for these proteins were validated, and functional cell-based assays were carried out to establish the role of each protein in their respective clone. MARCKS and Desmoglein-3 were found to reduce cell motility in vitro upon transient protein knockdown using siRNA. Coimmunoprecipitation of Desmoglein-3 revealed a potential interaction with mitochondrial proteins, a novel finding in lung cancer. Shootin-1, a relatively uncharacterised protein, was also found to reduce cell motility upon protein knockdown. Shootin-1 was also found to have novel potential binding partners, such as Semenogelin-1. Shootin-1 and Desmoglein-3 expression were affected by co-culture of the clonal subpopulations, highlighting the heterogeneous nature of the original DLKP cell line and the influences this has on protein expression.