posted on 2021-12-09, 16:33authored byErika
N. Cline, Carina Alvarez, Jiana Duan, Steven M. Patrie
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.