Online μSEC²-nRPLC-MS for Improved Sensitivity of Intact Protein Detection of IEF-Separated Nonhuman Primate Cerebrospinal Fluid Proteins

Abstract

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 (μSEC 2 -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 onedimensional nRPLC-MS with approximately 100× less CSF than previous OFFGEL studies. Furthermore, the modular design of the μSEC 2 -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.

Chromatographic separation of peptides and proteins for characterization of proteomes

“…195 Such offline two-dimensional (2D) SEC-RPLC allowed for the detection of more than 4000 unique proteoforms with M w up to 223 kDa and a 15-fold increase in the detection of proteins above 60 kDa, compared to one-dimensional RPLC. Patrie et al developed the online SEC-RPLC-MS platform to analysis of intact proteins from cerebrospinal fluid, 196 which was robust and promising for large-scale top-down analysis. Wu et al developed offline two-dimensional separation using high-pH and low-pH RPLC for characterizing proteoforms from E. coli 197 and human HeLa cells.…”

Recent stationary phase‐based fractionation strategies in proteomic analysis

“…A study on the efficiency of removal of low MW detergents (Triton X‐100 or CHAPS) by the first SEC column showed an increase in 95% of the detection of proteins of interest in comparison with the no‐filter control. When both SEC columns were operated together with nano RPLC‐MS, the signal to noise of low molecular interferents was decreased 20.5 times, along with a 34.4% decrease in the detection of high MW intact mass tags, which is in the range of the large interferents [53].…”

Small-Scale Serial Size Exclusion Chromatography (s3SEC) for High Sensitivity Top-Down Proteomics of Large Proteoforms

“…To overcome these limitations, size-based separations have typically been employed to isolate the high MW species from the low MW species. − Although gel-based techniques , provide good separation based on MW, the use of sodium dodecyl sulfate (SDS) requires significant sample cleanup prior to MS analysis, resulting in reduced throughput and protein recovery. , We have previously shown the advantages of using liquid chromatography, specifically size exclusion chromatography (SEC), as a suitable alternative for size-based intact protein separation. − Having the flexibility to use a wide range of MS-compatible mobile phases without the need for surfactant removal is highly advantageous when coupling offline fractionation to direct infusion or a second dimension of separation. , However, SEC using wider column diameters (i.e., 9.4 mm i.d.) has limitations, including large sample requirements and postfractionation sample handling (sample pooling and/or concentration), preventing its application to sample-limited systems.…”