Cellular heterogeneity of solid tumors represents a common problem in mass spectrometry (MS)-based analysis of tissue specimens. Combining immuno-laser capture microdissection (iLCM) and mass spectrometry (MS) provides a means to study proteins that are specific for pure cell subpopulations in complex tissues. CD24, as a cell surface marker for detecting pancreatic cancer stem cells (CSCs), is directly correlated with the development and metastasis of pancreatic cancer. Herein, we describe an in-depth proteomic profiling of frozen pancreatic CD24(+) adenocarcinoma cells from early stage tumors using iLCM and LC-MS/MS and a comparison with CD24(-) cells dissected from patient-matched adjacent normal tissues. Approximately 40 nL of tissue was procured from each specimen and subjected to tandem MS analysis in triplicate. A total of 2665 proteins were identified, with 375 proteins in common that were significantly differentially expressed in CD24(+) versus CD24(-) cells by at least a 2-fold change. The major groups of the differentially overexpressed proteins are involved in promoting tumor cell migration and invasion, immune escape, and tumor progression. Three selected candidates relevant to mediating immune escape, CD59, CD70, and CD74, and a tumor promoter, TGFBI, were further validated by immunohistochemistry analysis on tissue microarrays. These proteins showed significantly increased expression in a large group of clinical pancreatic adenocarcinomas but were negative in all normal pancreas samples. The significant coexpression of these proteins with CD24 suggests that they may play important roles in the progression of pancreatic cancer and could serve as promising prognosis markers and novel therapeutic targets for this deadly disease.