Multidimensional proteomics of human serum using parallel chromatography of native constituents and microplate technology

Abstract

A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags.

Mass spectrometry of peptides and proteins from human blood

“…Schizophrenia was quantitatively compared to control samples at the level of digested peptides (Levin et al, 2007;Levin et al, 2009). Multidimensional chromatography identified 32,288 peptides matching 2,669 serum proteins and revealed that alpha1B-glycoprotein, cysteine-rich secretory protein and an apparently low molecular-weight albumin were markers of severe inflammation (Horn et al, 2006;Rozet et al, 2006;Baum et al, 2008;Kreusch et al, 2008).…”

Recent advances in proteolysis and peptide/protein separation by chromatographic strategies

“…However, much progress has been made in this area. Horn et al [49] presented a high-throughput procedure for proteome research of native constituents. Human serum sample was first separated with SEC column in a mild condition and collected in a 96-well microplate.…”

Searching biomarker candidates in serum using multidimensional native chromatographyI. Enhanced separation method

“…The first separation step and analysis of the resulting fractions are described in [11]. Briefly, a mixture of 0.9 mL serum and 0.1 mL 10 mM Tris/HCl, pH 7.4, containing 150 mM NaCl was loaded onto a HiLoad Superdex TM 200 column (16/60, Pharmacia, Uppsala, Sweden) equilibrated with 10 mM Tris/HCl, pH 7.4, containing 150 mM NaCl.…”

“…This method was adapted to human serum for searching indestructible biomarker candidates within blood samples, whereas plasma is generally preferred to serum for in-depth proteome analysis [12]. The separation protocol of [11] was improved with respect to resolution, throughput, reproducibility, robustness, and simplicity. The present paper shows the characteristics of the improved method whereas the accompanying paper describes application of the search principle to model diseases.…”

“…A native separation technique combining two different chromatographic techniques orthogonally was developed in our group [11]. The technique is compatible with microplate technology and yields liquid fractions with proteins that exhibit defined small ranges of characteristic parameters, such as size or charge.…”