AUTOMATED NATIVE SAMPLE PREPARATION

A multidimensional chromatographic method is presented which is adapted for fractionation of body fluids for proteomic analysis and biomarker search. This method combines native size exclusion (SEC, first dimension, 1D), followed by anion exchange (AEC, 2D) and lectin affinity (LAC, 3D) chromatography. After serial 1D-fractionation, all further procedures are performed in microplate format. Thus, beginning with 2D-fractionation, parallelization and automation is achieved for separation, spectrophotometric readout, temporary storage, hit picking, medium exchange, digest, desalting, and finally storage within an autosampler of LC-MS. 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 adapted software packages. Some tools and tricks have been developed in order to improve sample preparation between fractionation steps, such as for medium exchange and desalting, re-concentration, and readout in microplates. Per working day, four serial 1D-runs or four synchronic 2D-runs can be performed including spectrophotometric readout. Thus, up to four samples may be processed comparably (1D) or in parallel (2D) within two days. Thereafter the majority of fractions are run through tryptic digest and mass spec; the few highly complex fractions remaining must be further separated by LAC before digest. Using commercially available columns (1D), the protein input of samples is scalable from 2 to more than 100 mg and from 2 to 1000 kDa molecular weights. Globally, protein recoveries based on UV measurements from the corresponding load are 93.7 ± 2.7, 97.1 ± 11.2, and 88.8 ± 3.6% and best precisions are 3.3, 3.8, and 5.1% CV with 1D-, 2D-, and 3D-fractionation, respectively. For process control and all-round analytics, a new software tool was developed accompanying the whole fractionation process. Its automatic interfaces collect entire data sets available including sample specification, location, and readout data of all fractions and identified proteins into a central data warehouse. Thus, immediate data access, comparison, and visualization in versatile charts and reports are possible. Besides automation, this method has several advantages: high proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, optional enzymatic and immunological analytics additional to mass spectrometry. Without exception, all intrinsic components and information are preserved after fractionation, including natural complex formation, fragmentation, and biological activities. Such information is beneficial for comprehensive profiling, for biomarker search, as well as for efficient evaluation. Preliminary applications show versatility with profiling plasma proteomes of humans, cattle, goat and mouse, and human cerebrospinal fluid (CSF). E.g., with two human plasma samples, 3166 and 816 non-redundant proteins could be identified supported by ≥1 and ≥2 peptides, respectively. Automated Native Sample Preparation for Proteome Analysis 3 Applying this method to biomarker search yields candidate markers that have already been validated in severe inflammation and Alport syndrome. Further application in neurodegenerative diseases and Psoriatic arthritis identifies huge numbers of altered proteins in CSF and plasma, respectively, which may present promising candidates for evaluation.