Category: Advances in Bioanalytics and Biomarkers

1231-B - Automated native sample preparation for reliable analysis of proteoforms of biomarkers

Monday, February 5, 2018
5:00 PM - 6:00 PM

Our automated, fast and precise method for 384-well microplates based proteome analysis and biomarker search was introduced on SLAS2015. Here the suitability of the method is demonstrated to identify urgently required disease typical proteoforms.

The central unit is an automated workstation enabling automated multichannel pipetting and robotic handling of microplates, reservoirs, and column arrays. The whole fractionation process, hit picking and analytics are controlled by a dedicated software package. Besides walkaway, this method has several advantages: high proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, outstanding precision and protein recovery, and optional enzymatic and immunological analytics in addition to mass spectrometry[1].

The multidimensional chromatographic method developed is adapted for the native pre-fractionation of proteins of body fluids. This method combines native size exclusion (SEC, first dimension, 1D), followed by anion exchange (AEC, 2D) and lectin affinity (LAC, 3D) chromatography. Beginning with 2D-fractionation, parallelization and automation is achieved for separation, spectrophotometric readout, temporary storage, hit picking, media exchange, digest, desalting, and finally storage within the autosampler of LC-MS.

Applications show versatility with profiling plasma proteomes of humans, cattle, goat and mouse, and human cerebrospinal fluid (CSF). E.g., with 12 human plasma samples, cumulatively 4997 and 816 non-redundant proteins could be identified supported by ≥1 and ≥2 peptides, respectively. The corresponding numbers are 5185 and 1691 with concentrated CSF from 51 individuals, 2263 and 507 with dog serum from 6 individuals, and 2542 and 343 with mouse serum from 18 individuals.

Applying this method to biomarker search yields altered protein concentrations of candidate markers that have already been validated in severe inflammation, Alport syndrome, psoriasis, and ALS[2][3][4][5][6][7].

Moreover, applying the principle of native protein separation, the analysis of body fluids uncovers various different chromatographic clusters of most of proteins. Currently, as supposed these clusters actually have been identified as characteristic differentially modified proteoforms.

Here, some examples of typical plasma proteins and biomarker candidates are given indicating the capabilities of the method to identify proteoforms exhibiting various posttranslational modifications directly from chromatographic subfractions.

[1] Wendler et al., in: Automation Systems of the 21st Century. Nova Science Publishers, NY, 2013, ISBN: 978-1-62948-262-0, Chapter 1, 1-50, open access

[2] Baum et al., J. Chromatogr. B, 2008, 876, 31-40,

[3] Hallbauer et al., Proteomics Clin Appl. 2010 Dec;4(12):953-61

[4] Pohl et al., Ped. Nephrology, 2013, 28, 2117-2123

[5] Muckova et al., J. Prot. Res. 14 (2015) 5202-14

[6] Reindl et al., J. Proteomics, 2016, 140, 55-61

[7] Prell et al., submitted

Heiko Oehme

Research & Technology Development
Analytik Jena AG, Jena, Germany
Jena, Thuringen, Germany

Research & Technology Development, Product Development, Digital Solutions