The pollen tube is a tip-growing system that delivers sperm to the ovule and thus is essential for sexual plant reproduction. Sucrose and other microelements act as nutrients and signaling molecules through pathways that are not yet fully understood. Taking advantage of high-throughput liquid chromatography coupled to mass spectrometry (LC-MS), we performed a label-free shotgun proteomic analysis of pollen in response to nutrient limitation using mass accuracy precursor alignment. We compared 168 LC-MS analyses and more than 1 million precursor ions and could define the proteomic phenotypes of pollen under different conditions. In total, 166 proteins and 42 phosphoproteins were identified as differentially regulated. These proteins are involved in a variety of signaling pathways, providing new insights into the multifaceted mechanism of nutrient function. The phosphorylation of proteins involved in cytoskeleton dynamics was found to be specifically responsive to Ca2+ and sucrose deficiency, suggesting that sucrose and extracellular Ca2+ influx are necessary for the maintenance of cytoskeleton polymerization. Sucrose limitation leads to widespread accumulation of proteins involved in carbohydrate metabolism and protein degradation. This highlights the wide range of metabolic and cellular processes that are modulated by sucrose but complicates dissection of the signaling pathways.