Multijunction Capillary Isoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables Isoelectric Point Fractionation of Intact Human Plasma Proteins for Biomarker Discovery

Abstract

Prefractionation of proteins is often employed to improve analysis specificity in proteomics. Prefractionation based on the isoelectric point (pI) is particularly attractive because pI is a well-defined parameter and it is orthogonal to hydrophobicity on which reversed-phase chromatography is based. However, direct capillary electrophoresis of blood proteins is challenging due to its high content of salts and charged small molecules. Here, we couple an online desalinator device to our multijunction capillary isoelectric focusing (MJ-CIEF) instrument and perform direct isoelectric separation of human blood plasma. In a proof-of-principle experiment, pooled samples of patients with progressive mild cognitive impairment and corresponding healthy controls were investigated. Injection of 3 μL of plasma containing over 100 μg of proteins into the desalinator was followed by pI fractionation with MJ-CIEF in less than 1 h. Shotgun proteomics of 12 collected fractions from each of the 5 replicates of pooled samples resulted in the identification and accurate quantification (median CV between the replicates is <4%) of nearly 365 protein groups from 4030 unique peptides (with <1% FDR for both peptides and proteins). The obtained results include several proteins previously reported as AD markers. The isoelectric point of each quantified protein was calculated using a set of 7 synthetic peptides spiked into the samples. Several proteins with a significant pI shift between their isoforms in the patient and control samples were identified. The presented method is straightforward, robust, and scalable; therefore, it can be used in both biological and clinical applications.

The deubiquitinase inhibitor b-AP15 induces strong proteotoxic stress and mitochondrial damage

“…Orthogonal fractionation of a sample reduces the sample complexity, which allows one to take deeper snapshots of the proteome, reaching more than 10,000 proteins (Sabatier, Saei et al) (paper not included in this thesis). Extensively used fractionation techniques include high pH reversed-phase chromatography (Batth, Francavilla et al 2014) and isoelectric focusing (Branca, Orre et al 2014;Pirmoradian, Astorga-Wells et al 2015).…”

Isoelectric point region pI≈7.4 as a treasure island of abnormal proteoforms in blood

“…3 µL of each sample were loaded into the MJ-CIEF device. Desalting was performed by buffer exchange in the online desalinator 18 using a washing solution composed of 0.5% Pharmalyte 6.6-7.6, 5% glycerol and 5mM dithiothreitol (DTT) in Milli-Q water at the flow rate of 500 µL/min for 10 min. Thereafter, the sample was transferred for pI separation into the capillary isoelectric focusing column.…”

“…Focused fractions were then mobilized and collected at the flow rate of 0.5 µL/min. A threetime stepwise releasing and refocusing was applied as described in detail previously 18 . For each sample, 20 fractions were collected.…”

“…Recently we have introduced a multijunction capillary isoelectric focusing (MJ-CIEF) device that combines high sample capacity (up to 100 µg), ease and speed of operation (<1 h) with low sample losses (<10%) 17,18 . This device helped to increase the depth of the proteome analysis of tryptic peptide mixtures 17 and blood plasma proteins 18 . Here we employed the MJ-CIEF fractionator for testing the pI-pH hypothesis in serum of patients with neurodegenerative disorders.…”

“…One of the reasons for proteoforms to appear in that forbidden region is aggregation, a process characteristic for AD as well as other neurodegenerative disorders 22 . The novel MJ-CIEF device is applied in the current study together with the optimized proteomics techniques developed previously in our lab 17,18,23 . For testing the pI-pH hypothesis, patients from Western Norway diagnosed with probable AD and matched for gender (female) and age (77±6 years) were selected 24 .…”