Mats Leeman (Presenter)
SOLVE Research and Consultancy AB
Bio: Senior scientist at SOLVE Research and Consultancy AB. Mats holds a PhD in Technical Analytical Chemistry from Lund Institute of Technology. He has been using AF4 since more than 15 years for various applications within pharmaceutical, specialty chemicals and biotech industries.
Authorship: Leeman, M. (1), Scott, B. (2), Lang, B. (3), Storm, MU.(1), Runyon, R. (1), Wiese, C. (2), Poetz, O. (3), Joos TO (3)
(1) SOLVE Research and Consultancy AB, Lund, Sweden, (2) Wellspring Clinical Lab Inc., Altamonte, FL, (3) NMI Natural and Medical Sciences Institute, Reutlingen, Germany
| Short Abstract A novel method integrating solid-phase free immunocomplex isolation for LC/MS based immunoassay approaches was established. Usually, LC/MS based immunoassays include an immunoprecipitation step capturing the endogenous and isotopic labelled reference peptides with antibodies conjugated to solid surfaces in bead or column format prior to MS analysis. Here, we present a method where the antibody-peptide complex is not precipitated, but isolated from plasma digest peptides by asymmetric field flow fractionation (AFFF). The use of AFFF allowed a solid-phase free immunocomplex enrichment from plasma digest, thereby avoiding contamination caused by high abundant peptides through solid-phase interaction with carrier surfaces and allowing the detection of C-reactive protein. |
Long Abstract
Introduction. The vast dynamic range of the plasma proteome (spanning more than 10 orders of magnitude) has chronically plagued its analysis and exploration. The massive analytical power of mass spectrometry continues to be challenged when exploring low abundance proteins by this massive dynamic range. State of the art methods such as SISCAPA that report enrichment of low abundance proteins up to 100,000 times merely reduce the complexity for proteins like cardiac troponin from 1 protein for every 10 billion albumins, to 1 protein for every 100,000 albumins.
With such complexity, ionization suppression characteristic of mass spectrometry makes it highly difficult to analyze low abundance proteins of interest. While liquid chromatography is useful, the narrow elution peaks of the low abundance proteins of interest continue to be overwhelmed by the wide elution peaks associated with high abundance proteins in plasma.
Immunoaffinity isolation of surrogate peptides along with stable isotope internal standards of plasma proteome digests is a useful method for quantifying plasma proteins with LC/MS. Typical isolation methods employ solid surfaces on which to immobilize such immunocomplexes. Non-specific binding of high abundance residues to isolation surfaces has been largely implicated as the source of high abundance residues in the isolated fractions.
We have demonstrated in the past that high abundance residues in isolated fractions can be largely avoided by eliminating immobilizing surfaces using ultracentrifugation to isolate the immunocomplex fraction (~155kDa) from the peptide digest fraction (< 5kDa). While demonstrating proof of concept, ultracentrifugation has not been shown to be readily production capable. We have now demonstrated avoiding high abundance residues in isolated fractions using a hybrid separation method that combines AFFF with ultrafiltration to isolate the immunocomplex fraction from the peptide digest fraction that is readily production capable.
Methods. We employ an asymmetric field flow fractionation channel in normal field flow mode using a 30kDa MCWO ultrafiltration membrane. In this configuration, the immunocomplex fraction is isolated by 1) permeation of the peptides across the membrane and 2) lower migration velocity across the channel.
The conditions for performing the separation are:
Sample: 1 ul human plasma digest
Stable isotopic internal standard: 5 pmol GYSIFSYATK
Antibody: 5 ug anti GYSIFSYATK
Incubation: 60 minutes mixing
Carrier solvent: Phosphate buffered solution + 3mM sodium azide
FFF Channel: Wyatt SC
Membrane: Regenerated cellulose 30kDa MWCO
Spacer: 490 um, narrow
Carrier flow rate: 1.0 ml/minute
Crossflow flow rate: 0.5 ml/minute
Output port flow rate: 0.5 ml/minute
Injection time: 3 minutes/0.2 ml/minut
Focusing time: 7 minutes
Immunocomplex fraction elution start time: 4.5 minutes
Immunocomplex fraction elution end time: 7.5 minutes
Immunocomplex fraction volume: 1.5 ml
The isolated fraction was concentrated by magnetic bead solid phase extraction. The concentrated fraction was injected into LC/MS for analysis.
Results. The isolated fraction was analyzed by LC/MS for presence of high abundance protein residues. 19 protein residues were identified. None of the identified residues are associated with high abundance plasma proteins. The identified residues were associated with antibody development. The signals associated with the identified proteins were much lower than the signals associated with the surrogate peptides.
Conclusions. The demonstrated isolation method is useful for LC/MS low-abundance plasma protein assays. Assay development is streamlined by avoiding complexities associated with incomplete isolation from digest residues. Higher sensitivity should be achievable by avoidance of ionization suppression from high abundance residues. Elution from LC should be cleaner due to avoidance of wide high abundance elution peaks.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Wellspring |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |