Nano-flow chromatography-tandem mass spectrometer (nLC-MS/MS) is a popular choice in proteome research as it provided high-sensitivity analysis with minimal sample usage, but its quantitative proteomics applications become restricted by low analytical throughput, and low robustness. To circumvent this limitation, we describe the performance of the new Vacuum Insulated Probe Heated ElectroSpray Ionization source (VIP-HESI) coupled to the Bruker timsTOF mass spectrometer and show it enhances micro-spray flow rate chromatography signals in comparison to nanospray source conditions using the CaptiveSpray (CS) and ElectroSpray Ionization (ESI) sources. In addition, using a 0.5 x 200mm and 1 × 150 mm columns, achieved excellent reproducibility of chromatographic retention time, coefficient of variation, and precision quantification using data from un-depleted mouse plasma, HeLa and K562 digest peptide samples.