Proteoform-resolved information, obtained by top–down (TD) “intact protein” proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and, in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry (MS) analysis of intact proteins in complex biological samples is hindered by the high dynamic range in protein concentration and mass, protein instability, and buffer complexity. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size-exclusion chromatography with nanoflow reversed-phase liquid chromatography and MS (μSEC2-nRPLC-MS). Online serial high-/low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges (5–155 kDa) prior to nRPLC-MS. The coupling of μSEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns, pre- and post-μSEC columns, to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application of the platform to cerebrospinal fluid (CSF) prefractionated by OFFGEL isoelectric focusing drastically increases the number of intact mass tags (IMTs) detected within the target mass range (5–30 kDa) in comparison to one-dimensional nRPLC-MS with approximately 100× less CSF than previous OFFGEL studies. Furthermore, the modular design of the μSEC2-nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.
ASJC Scopus subject areas
- Analytical Chemistry