Mass spectrometry-based analysis of trace analytes, such as the single cell proteome, benefit from operation at low flow rates (i.e., <50 nL/min). However, the standard high-pressure binary pumps needed to achieve these flow rates are not commercially available, requiring splitting of the gradient flow to achieve low-nanoliter-per-minute flow rates, which wastes solvent and can lead to flow inconsistencies. To address this, we have created a new method for creating gradients by combining plugs of mobile phase of increasing solvent strength together in a column and then using Taylor dispersion to form the desired smooth gradient profile. Additionally, our method dramatically reduces costs, as only a single isocratic high-pressure pump is required. Following optimization of gradient shapes using UV absorption for both 10- and 20-min active gradients, we measured 200 pg injections of HeLa digest on a timsTOF Ultra mass spectrometer in DIA mode. We then investigated differences in protein expression between single cells originating from two different colonies of ATG-knockout HeLa cells. Thousands of proteins were quantified, and a potential mechanism explaining differential immune responses of these two colonies upon exposure to viral DNA treatment was determined.