Looseness is a defect found in leather that reduces its quality by causing a wrinkly appearance in the finished product, resulting in a reduction in its value. Earlier studies on loose leather using microscopy and Raman spectroscopy reported a change in the collagen structure of loose leather. In this study, proteomics was used to investigate the possible molecular causes of looseness in the raw material, the first time such a study has been carried out. Proteins extracted from two regions of raw hide using two different methods were analysed; those taken from the distal axilla, an area prone to looseness, and those taken from the backbone which is less prone to looseness. Analyses using 1DE-LC-MS/MS showed that although the overall collagen concentration was similar in both areas of the hide, the distribution of the different types of collagen differed. Specifically, concentrations of type I collagen, and the collagen-associated proteoglycan decorin were lower in samples taken from the distal axilla, symptomatic of a collagen network with excess space seen for these samples using confocal microscopy. This study suggests a possible link between the molecular components of raw cattle hide and looseness and more importantly between the molecular components of skin and skin defects. There is therefore potential to develop biomarkers for looseness which will enable early preventative action.