bottom-up-shotgun-proteomics-to-identify-comprehensive-cochlear

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

University of South Florida

Mass spectrometry was used in conjunction with gel electrophoresis and liquid chromatography, to determine peptide sequences from American alligator (Alligator mississippiensis) leukocytes and to identify similar proteins based on homology. The goal of the study was to generate an initial database of proteins related to the alligator immune system. We have adopted a typical proteomics approach for this study. Proteins from leukocyte extracts were separated using two-dimensional gel electrophoresis and the major bands were excised, digested and analyzed by on-line nano-LC MS/MS to generate peptide sequences. The sequences generated were used to identify proteins and characterize their functions. The protein identity and characterization of the protein function were based on matching two or more peptides to the same protein by searching against the NCBI database using MASCOT and Basic Local Alignment Search Tool (BLAST). For those proteins with only one peptide matching, the phylum of the matched protein was considered. Forty-three proteins were identified that exhibit sequence similarities to proteins from other vertebrates. Proteins related to the cytoskeletal system were the most abundant proteins identified. These proteins are known to regulate cell mobility and phagocytosis. Several other peptides were matched to proteins that potentially have immune-related function.

Proteome analysis of the leukocytes from the American alligator (Alligator mississippiensis) using mass spectrometry.

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35kDa protein was ~98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis).

Proteomic analysis of sensory organs such as the cochlea is challenging due to its small size and difficulties with membrane protein isolation. Mass spectrometry in conjunction with separation methods can provide a more comprehensive proteome, because of the ability to enrich protein samples, detect hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. GELFrEE as well as different separation and digestion techniques were combined with FASP and nanoLC-MS/MS to obtain an in-depth proteome analysis of cochlear sensory epithelium from 30-day-old mice. Digestion with LysC/trypsin followed by SCX fractionation and multiple nanoLC-MS/MS analyses identified 3773 proteins with a 1% FDR. Of these, 694 protein IDs were in the plasmalemma. Protein IDs obtained by combining outcomes from GELFrEE/LysC/trypsin with GELFrEE/trypsin/trypsin generated 2779 proteins, of which 606 additional proteins were identified using the GELFrEE/LysC/trypsin approach. Combining results from the different techniques resulted in a total of 4620 IDs, including a number of previously unreported proteins. GO analyses showed high expression of binding and catalytic proteins as well as proteins associated with metabolism. The results show that the application of multiple techniques is needed to provide an exhaustive proteome of the cochlear sensory epithelium that includes many membrane proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000231.

Article	Year
Proteome analysis of the leukocytes from the American alligator (Alligator mississippiensis) using mass spectrometry. Comparative biochemistry and physiology. Part D, Genomics & proteomics\| PubMed ID: 20920849	2010
Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis). Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology\| PubMed ID: 22085437	2012
In-depth proteomic analysis of mouse cochlear sensory epithelium by mass spectrometry. Journal of proteome research\| PubMed ID: 23721421	2013

Lancia N.F. Darville

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