PPLine: An Automated Pipeline for SNP, SAP, and Splice Variant Detection in the Context of Proteogenomics.

The fundamental mission of the Chromosome-Centric Human Proteome Project (C-HPP) is the research of human proteome diversity, including rare variants. Liver tissues, HepG2 cells, and plasma were selected as one of the major objects for C-HPP studies. The proteogenomic approach, a recently introduced technique, is a powerful method for predicting and validating proteoforms coming from alternative splicing, mutations, and transcript editing. We developed PPLine, a Python-based proteogenomic pipeline providing automated single-amino-acid polymorphism (SAP), indel, and alternative-spliced-variants discovery based on raw transcriptome and exome sequence data, single-nucleotide polymorphism (SNP) annotation and filtration, and the prediction of proteotypic peptides (available at https://sourceforge.net/projects/ppline). In this work, we performed deep transcriptome sequencing of HepG2 cells and liver tissues using two platforms: Illumina HiSeq and Applied Biosystems SOLiD. Using PPLine, we revealed 7756 SAP and indels for HepG2 cells and liver (including 659 variants nonannotated in dbSNP). We found 17 indels in transcripts associated with the translation of alternate reading frames (ARF) longer than 300 bp. The ARF products of two genes, SLMO1 and TMEM8A, demonstrate signatures of caspase-binding domain and Gcn5-related N-acetyltransferase. Alternative splicing analysis predicted novel proteoforms encoded by 203 (liver) and 475 (HepG2) genes according to both Illumina and SOLiD data. The results of the present work represent a basis for subsequent proteomic studies by the C-HPP consortium.

Registration of the protein with compact disk.

CD-based optico-acoustical biosensor (OAB) was used for detection of various types of proteins represented by bovine serum albumin (BSA), heme-containing myoglobin (Mb), monoclonal antibody against viral protein marker of hepatitis B (anti-HBsAg) and membrane-bound cytochrome P450scc (P450scc). We applied standard compact disc reader (CD-ROM) as an optical analyzer and a standard compact disc (CD) as a biochip containing immobilized protein molecules. This biosensor can translate into a digital code the changes of optical signal from the proteins and their complexes immobilized on the CD surface. Then, the digital code is translated into an acoustic series or, in other words, into a "music of proteins". We demonstrate the use of the OAB for direct detection of proteins with different molecular weights, such as BSA, Mb, P450scc, anti-HBsAg with the concentration detection limit (DL) about 10(-7)M. By signal amplification achieved with autometallography, a higher sensitivity level (DL∼10(-9)M) for the detection of myoglobin was obtained. The method of OAB-detection of proteins is cheap: it requires no special equipment like spectrometers, refractometers and other devices. Due to the fact that acoustic series of the protein complexes antigen/antibody differs from that of single proteins, the OAB-detection is of particular interest for rapid assay in yes/no data type and for home diagnostics. Combination of the OAB with a mass spectrometer allowed the detection and identification of the target proteins fished out directly onto a standard CD surface.

AFM fishing nanotechnology is the way to reverse the Avogadro number in proteomics.

Future development of proteomics may be hindered by limitations in the concentration sensitivity of widespread technological approaches. The concentration sensitivity limit (CSL) of currently used approaches, like 2-DE/LC separation coupled with MS detection, etc., varies from 10(-9) to 10(-12) M. Therefore, proteomic technologies enable detection of up to 20% of the protein species present in the plasma. New technologies, like atomic force microscopy (AFM molecular detector), enable the counting of single molecules, whereas biospecific fishing can be used to capture these molecules from the biomaterial. At the same time, fishing also has thermodynamic limitations due to the reversibility of the binding. In cases where the fishing becomes irreversible, its combination with an AFM detector enables the registration of single protein molecules, and that opens up a way to lower the CSL down to the reverse Avogadro number.