Next Generation Proteomic Pipeline for Chromosome-Based Proteomic Research Using NeXtProt and GENCODE Databases

Abstract

Human Proteome Project aims to map all human proteins including missing proteins as well as proteoforms with post translational modifications, alternative splicing variants (ASVs), and single amino acid variants (SAAVs). neXtProt and Ensemble databases are usually used to provide curated information on human coding genes. However, to find these proteoforms, we (Chr #11 team) first introduce a streamlined pipeline using customized and concatenated neXtProt and GENCODE originated from Ensemble, with controlled false discovery rate (FDR). Because of large sized databases used in this pipeline, we found more stringent FDR filtering (0.1% at the peptide level and 1% at the protein level) to claim novel findings, such as GENCODE ASVs and missing proteins, from human hippocampus data set (MSV000081385) and ProteomeXchange (PXD007166). Using our next generation proteomic pipeline (nextPP) with neXtProt and GENCODE databases, two missing proteins such as activity-regulated cytoskeleton-associated protein (ARC, Chr 8) and glutamate receptor ionotropic, kainite 5 (GRIK5, Chr 19) were additionally identified with two or more unique peptides from human brain tissues. Additionally, by applying the pipeline to human brain related data sets such as cortex (PXD000067 and PXD000561), spinal cord, and fetal brain (PXD000561), seven GENCODE ASVs such as ACTN4-012 (Chr.19), DPYSL2-005 (Chr.8), MPRIP-003 (Chr.17), NCAM1-013 (Chr.11), EPB41L1-017 (Chr.20), AGAP1-004 (Chr.2), and CPNE5-005 (Chr.6) were identified from two or more data sets. The identified peptides of GENCODE ASVs were mapped onto novel exon insertions, alternative translations at 5'-untranslated region, or novel protein coding sequence. Applying the pipeline to male reproductive organ related data sets, 52 GENCODE ASVs were identified from two testis (PXD000561 and PXD002179) and a spermatozoa (PXD003947) data sets. Four out of 52 GENCODE ASVs such as RAB11FIP5-008 (Chr. 2), RP13-347D8.7-001 (Chr. X), PRDX4-002 (Chr. X), and RP11-666A8.13-001 (Chr. 17) were identified in all of the three samples.

The origin of the expressed retrotransposed gene ACTBL2 and its influence on human melanoma cells’ motility and focal adhesion formation

“…These findings suggest that actbl2 undergoes similar post-translational modifications as other actin isoforms 40 . Actbl2 unique peptides were detected both in healthy tissues 23,33,36,41 or cell lines 21,34,38 , and in tumor tissues [30][31][32]39,42 or cell lines 21,22,24,26,28,29,35,43 .…”

Quantitative Analysis of Antibody Survival across the Infant Digestive Tract Using Mass Spectrometry with Parallel Reaction Monitoring

“…Of the ten tryptic peptides from the known palivizumab VL and VH regions, only two peptides, LLIYDTSK and VTNMDPADTATYYCAR (with carbamidomethylation), were detected across the entire concentration range. Next, we identified whether the peptide sequences observed in MS analysis were unique using the peptide uniqueness checker in neXtProt [ 36 ]. This analysis revealed that seven out of ten peptides did not match any sequences in the human protein database.…”

Proteogenomics and its applications in biology and precision medicine

“…Nhóm nghiên cứu Chromosome-11 trong dự án C-HPP đã ghép nối cơ sở dữ liệu NeXtProt (https://www.nextprot.org/) và GENCODE (https://www.gencodegenes.org/) để phân tích các bộ dữ liệu MS từ các mô não, vỏ não, tủy sống, não thai nhi, tinh hoàn và tinh trùng. Họ đã xác định các protein bị thiếu và các biến thể ghép nối thay thế (ASV, alternative spliced variants) của GENCODE ở các phần chèn exon mới, các bản dịch mã thay thế ở vùng 5' chưa được dịch mã hoặc trình tự mã hóa protein mới (Hwang et al, 2017 (Omenn et al, 2019).…”