Rothberg Returns: DNA Sequencing Legend Incites Protein Sequencing Revolution

2021 ◽  
Vol 41 (8) ◽  
pp. 10, 12
Author(s):  
Alex Philippidis
Keyword(s):  
Dna Sequencing ◽  
Protein Sequencing

scholarly journals Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

2022 ◽  
Author(s):  
Brian D Reed ◽  
Michael J Meyer ◽  
Valentin Abramzon ◽  
Omer Ad ◽  
Pat Adcock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Sequencing ◽  
Real Time ◽  
Single Molecule ◽  
Dynamic Range ◽  
Protein Sequencing ◽  
Peptide Sequence ◽  
Single Amino Acid ◽  
Functional Components

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

scholarly journals Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase: analysis by mass spectrometry and by protein and DNA sequencing

1996 ◽  
Vol 314 (3) ◽  
pp. 817-825 ◽  
Author(s):  
Robert HAAS ◽  
Brent C. JACKSON ◽  
Bruce REINHOLD ◽  
John D. FOSTER ◽  
Terrone L. ROSENBERRY
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Dna Sequencing ◽  
Protein Sequencing ◽  
Peptide Sequence ◽  
Partial Reduction ◽  
Bovine Erythrocyte ◽  
Gpi Anchor ◽  
Amino Acid Peptide ◽  
Fragmentation Patterns

Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-PO4-Hex-Hex-Hex(PO4-ethanolamine) (HexNAc)-HexN(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a …CTC… motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered.

Ribosomal proteins in halobacteria

1989 ◽  
Vol 35 (1) ◽  
pp. 195-199 ◽  
Author(s):  
Makoto Kimura ◽  
Evelyn Arndt ◽  
Tomomitsu Hatakeyama ◽  
Tamiko Hatakeyama ◽  
Junko Kimura
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequencing ◽  
Glutamic Acid ◽  
Ribosomal Proteins ◽  
Chemical Method ◽  
Amino Acid Sequences ◽  
Protein Sequencing ◽  
Arginine Residues

The amino acid sequences of 16 ribosomal proteins from archaebacterium Halobacterium marismortui have been determined by a direct protein chemical method. In addition, amino acid sequences of three proteins, S11, S18, and L25, have been established by DNA sequencing of their genes as well as by protein sequencing. Comparison of their sequences with those of ribosomal proteins from other organisms revealed that proteins S14, S16, S19, and L25 are related to both eukaryotic and eubacterial ribosomal proteins, being more homologous to eukaryotic than eubacterial counterparts, and proteins S12, S15, and L16 are related to only eukaryotic ribosomal proteins. Furthermore, some proteins are found to be similar to only eubacterial proteins, whereas other proteins show no homology to any other known ribosomal proteins. Comparisons of amino acid compositions between halophilic and nonhalophilic ribosomal proteins revealed that halophilic proteins gain asparatic and glutamic acid residues and significantly lose lysine and arginine residues. In addition, halophilic proteins seem to lose isoleucine as compared with Escherichia coli ribosomal proteins.Key words: halobacteria, ribosomal proteins, amino acid sequence.

Imaging polymers, proteins and dna in aqueous solutions with the atomic force microscope

1989 ◽  
Vol 47 ◽  
pp. 32-33
Author(s):  
S.A.C. Gould ◽  
B. Drake ◽  
C.B. Prater ◽  
A.L. Weisenhorn ◽  
S.M. Lindsay ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Sequencing ◽  
Aqueous Solutions ◽  
Atomic Force Microscope ◽  
Piezoelectric Crystal ◽  
Atomic Force ◽  
Structure Of Molecules ◽  
Optical Lever

The atomic force microscope (AFM) is an instrument that can be used to image many samples of interest in biology and medicine. Images of polymerized amino acids, polyalanine and polyphenylalanine demonstrate the potential of the AFM for revealing the structure of molecules. Images of the protein fibrinogen which agree with TEM images demonstrate that the AFM can provide topographical data on larger molecules. Finally, images of DNA suggest the AFM may soon provide an easier and faster technique for DNA sequencing.The AFM consists of a microfabricated SiO2 triangular shaped cantilever with a diamond tip affixed at the elbow to act as a probe. The sample is mounted on a electronically driven piezoelectric crystal. It is then placed in contact with the tip and scanned. The topography of the surface causes minute deflections in the 100 μm long cantilever which are detected using an optical lever.

TB OR NOT TB?… THAT IS THE QUESTION: A RARE MYCOBACTERIUM (M. INTERMEDIUM) IDENTIFIED BY DNA SEQUENCING

2001 ◽  
Vol 28 (10) ◽  
pp. 549-554
Author(s):  
Ryan N. Cole ◽  
Stewart W. West ◽  
Christine L. Terrell ◽  
Glenn D. Roberts ◽  
Iftikhar Ahmed
Keyword(s):  
Dna Sequencing

A brief history of (DNA sequencing) time

2007 ◽  
Vol 8 (S1) ◽  
pp. S21-S21
Author(s):  
Elaine R. Mardis
Keyword(s):  
Dna Sequencing ◽  
History Of

Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Meng-Yin Li ◽  
Jie Yang ◽  
Ya-Qian Wang ◽  
Xue-Yuan Wu ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Genetic Diseases ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Lesion Detection ◽  
The Novel ◽  
Structural Variations ◽  
Dna Lesion ◽  
Base Lesion

DNA lesion such as metholcytosine(<sup>m</sup>C), 8-OXO-guanine(<sup>O</sup>G), inosine(I) <i>etc</i> could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (<sup>m</sup>C, <sup>O</sup>G, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

Sign in / Sign up

Export Citation Format

Share Document