- Proteins have no base pairing and consequently there is no technique of protein amplification like the polymerase chain reaction (PCR), so detection is more difficult,
- The average protein concentration is 102–108 copies/cell
Methods of proteomics
| LC-MS | Dominant methods | referred to as bottom-up or shotgun proteomics, as for technical reasons proteins are digested, mostly with trypsin, cutting C-terminal to lysine and arginine, before MS analysis. The principle of LC–MS/MS analysis is that peptides from the enzyme digested sample are separated by LC → before being analysed by MS | |||||||||||||||||||||||||||||||||||||||||||||||||||||
| Somascan 각각의 RFU 값을 구하고 → 그 FC 구하고 (절대 FC네) → log2 구함 | https://somalogic.com/somascan-discovery/ SOMAmer = Slow Off-rate Modified Aptamer |
Attached please find the complete list of proteins currently in the SOMAScan 7k panel. I didn't see NLRP3 there but they might have it. As I understand it, the samples would cost $750 for the full 7k scan. There are also less expensive options. They can do off-the-shelf or custom-made panels of 1500 proteins for ~$350/sample. Below are some refs for brain and CSF SOMAScans they've run. I am currently trying to find a time for the SL rep to give a talk that is convenient for everyone. Thanks, -Shane https://www.nature.com/articles/s41593-021-00886-6 https://www.nature.com/articles/s41586-021-04183-x https://alzres.biomedcentral.com/articles/10.1186/s13195-017-0258-6 https://www.nature.com/articles/s41598-018-26237-3 SomaScan does not discriminate protein isoforms or post-translational modification (Kaiser 2023) The SomaScan Platform is enabled by the generation of protein-capture reagents called SOMAmer® (Slow Off-rate Modified Aptamer) reagents. SOMAmer reagents consist of short single-stranded DNA sequences that incorporate hydrophobic modifications, greatly expanding the physicochemical diversity of the large randomized nucleic acid libraries from which the SOMAmer reagents are selected. The readout in relative fluorescent units (RFU) is directly proportional to the amount of target epitope in the initial sample. But!!! (Bryda, 2013 #2613) "semiquantitative", 5000 개중 대부분의 protein 에 대해 standard curve x, but just relative changes in expression Issue: non-specificity (20190118 Somalogic's ~~. Pdf)
| |||||||||||||||||||||||||||||||||||||||||||||||||||||
| WB | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Olink | https://www.bioxpedia.com/olink-proteomics/ |
multiplex immunoassay-qPCR Immunoreaction → extension reaction → pre-amplification → detection → QC and generation of NPX proximity extension assay (PEA) is an emerging technology developed by Olink Proteomics (Uppsala, Sweden), which merges quantitative real-time PCR with multiplex immunoassays. The basis of PEA is a dual recognition of a targeted biomarker through a matched pair of antibodies labeled with unique DNA oligonucleotides. Here, biomarker-specific DNA "barcodes" are quantified by microfluidic qPCR allowing for high-throughput relative quantification of up to 1161 human plasma proteins using a few microliters of biofluids (1 µL for the quantification of 92 biomarkers). https://olink.com/resources-support/document-download-center/ (여기 various panels, mouse panel) 1. Immunoreaction: Allow the 92 antibody probe pairs to bind to their respective proteins in your samples 2. Extension and pre-amplification Extend and pre-amplify 92 unique DNA reporter sequences by proximity extension. Amplification and detection 3. Quantify each biomarker's DNA reporter using high throughput real-time qPCR. |
semi-quantitative, where changes in protein levels in one population are quantified relative to another. The assays generate quantitation cycle (Cq) values that are then normalized and converted to Olink's arbitrary Normalized Protein eXpression (NPX) unit, which can be used for further statistical analyses. NPX gives relative quantification. NPX is on a log2 scale. NPX should be compared between samples for each assay separately. Overlapping proteins between olink and somascan: ~40% ({Katz, 2022 #2629} Overlapping proteins between olink and LC/MS: ~10% {Petrera 2021 #2630} in plasma Olink recommends that assays with a large proportion of samples below LOD is excluded from the analysis. The limit for exclusion should be decided on a study basis and consider design including sample size and experimental variables. Suitable exclusion limits may be in the range of less than 25-50% of samples above LOD. | ||||||||||||||||||||||||||||||||||||||||||||||||||||
Database of Protein structure
| Proteopedia | http://proteopedia.org/wiki/index.php/Glucocerebrosidase | 3D |
| Open Targets | https://opentargets.onetakeda.com/target/ENSG00000094631 | |
| PDBe (Protein Data Bank in Europe) | https://www.ebi.ac.uk/pdbe/ | |
| Others | https://en.wikipedia.org/wiki/Protein_structure_database | |
| UNIPROT.ORG | uniprot.org | |
| GenBank | ||
| OMIM | ||
| Protein Data Bank (PDB) | ||
| ENZYME | ||
| Ensembl | ||
| Hugo Gene Nomenclature Committee (HGNC) | ||
| Mouse Genome Informatics (MGI) | ||
| Gene Ontology | ||
| Gene Expression Omnibus (GEO) | ||
| The Cancer Genome Atlas (TCGA) |
GFP (green fluorescent protein)
- a protein composed of 238 amino acid residues (26.9 kDa)
- exhibits bright green fluorescence when exposed to light, in the blue to ultraviolet range.[2][3]
- GFP gene is frequently used as a reporter of expression.[5]
Homology
Search tools: BLAST, HMMER and Infernal.[16] Blast는 님이 가지고 계시는 sequence가 무엇이랑 가장 가까운지를 알려주는 것입니다. 가장 확율이 높은것부터 순서대로 일정주를 보여주죠.
Homology search tools may take an individual nucleic acid or protein sequence as input, or use statistical models generated from multiple sequence alignments of known related sequences.
| 여러 종 한꺼번에 결과 나옴. (Better 인듯) https://www.uniprot.org/blast | 두 종 입력 https://blast.ncbi.nlm.nih.gov/BlastAlign.cgi# |
BLAST (uniprot.org/blast):
BLAST
⚠ Scheduled maintenance will cause BLAST service to be unavailable at the following time
5/7/2024, 8:00:00 AM to 5/8/2024, 8:00:00 AM (America/Los_Angeles time zone)
Please submit jobs before or after this period. In addition, running jobs will be interrupted and so will need to be resubmitted.
Find a protein sequence to run BLAST sequence similarity search by UniProt ID (e.g. P05067 or A4_HUMAN or UPI0000000001)
[ P14173 ] x ⓘ
OR
Enter one or more sequences (5 max). You may also load from a text file.
위 박스에 UniProt ID 같은 것 넣고 → Run BLAST 누름
BLAST Align (blast.ncbi.nlm.nih.gov/BlastAlign.cgi#):
Enter Query Sequence
Enter accession number(s), gi(s), or FASTA sequence(s) ⓘ Clear
[ D4A523 ]
Or, upload file: Choose File No file chosen
Job Title: D4A523 RecName: Full=NACHT, LRR and PYD dom...
Enter a descriptive title for your BLAST search ⓘ
☑ Align two or more sequences ⓘ
두 종의 UniProt ID 혹은 Gene ID 를 각각 위, 아래 box에 입력 → BLAST 누름
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Top wrap-around proteomics cell | (among which 5,880 proteins were quantified. → results: We detected 124 up-regulated proteins and 92 down-regulated proteins after perifosine treatment) | The cell header and the experimental design rows are on 20240722_184649; only the tail of the cell is visible. |
| WB row | (empty) | The Western Blot row body is empty in the source on this capture. |
| Olink schematic icons | (eye / DNA helix / sphere icons numbered 1–3) | Decorative numbered icons for Immunoreaction / Extension and pre-amplification / Amplification and detection; preserved as evidence only. |
| BLAST screenshot tail | (Job Title input continues below the cropped form) | Bottom of the BlastAlign form is cut at the Align two or more sequences checkbox; remaining form fields continue on 20240722_184656. |