scholarly journals ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

2020 ◽  
Vol 19 (12) ◽  
pp. 2139-2156
Author(s):  
Diana Samodova ◽  
Christopher M. Hosfield ◽  
Christian N. Cramer ◽  
Maria V. Giuli ◽  
Enrico Cappellini ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Protein Identification ◽  
De Novo ◽  
Human Cell Line ◽  
Bone Collagen ◽  
Sequence Coverage ◽  
Noncollagenous Proteins ◽  
High Coverage ◽  
Ancient Bone ◽  
Proline Rich Protein

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.

scholarly journals A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals chromosomal rearrangements in rainbow trout

2021 ◽  
Author(s):  
Guangtu Gao ◽  
Susana Magadan ◽  
Geoffrey C Waldbieser ◽  
Ramey C Youngblood ◽  
Paul A Wheeler ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Chromosome Number ◽  
Genome Assembly ◽  
De Novo Assembly ◽  
De Novo ◽  
Sequence Data ◽  
Structural Variations ◽  
High Coverage ◽  
Haploid Chromosome Number ◽  
Long Reads

Abstract Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2 N = 64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13.

Identification of α-enolase as a prognostic and diagnostic precancer biomarker in oral submucous fibrosis

2017 ◽  
Vol 71 (3) ◽  
pp. 228-238 ◽  
Author(s):  
Swarnendu Bag ◽  
Debabrata Dutta ◽  
Amrita Chaudhary ◽  
Bidhan Chandra Sing ◽  
Mousumi Pal ◽  
...  
Keyword(s):  
De Novo ◽  
Pain Treatment ◽  
Oral Submucous Fibrosis ◽  
Peptide Sequencing ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Sequence Coverage ◽  
Protein Marker ◽  
Peptide Mass ◽  
Submucous Fibrosis

AimsDiagnostic ambiguities regarding the malignant potentiality of oral submucous fibrosis (OSF), an oral precancerous condition having dysplastic and non-dysplastic isoforms are the major failure for early intervention of oral squamous cell carcinoma (OSCC) patients. Our goal is to identify proteomic signatures from biopsies that can be used as precancer diagnostic marker for patient suffering from OSF.MethodsThe high throughput techniques adopting de novo peptide sequencing (1D SDS-PAGE coupled nanoLC MALDI tandem mass spectrometry (MS/MS)-based peptide mass fingerprint), immunohistochemistry (IHC), Western blot (WB) and real-time PCR (RT-PCR) analysis are considered for such biomarker identification and multilevel validations.ResultsAlpha-enolase is identified as an overexpressed protein in biopsies of oral submucous fibrosis with dysplasia (OSFWD) compared with oral submucous fibrosis without dysplasia (OSFWT) and normal oral mucosa (NOM). Total proteome analysis of an overexpressed protein band around 47 kDa of OSFWD identifies 334 peptides corresponding to 61 human proteins. Among them α-enolase is identified as a prime protein with highest number of peptides (44 out of 334 peptides) and sequence coverage (66.4%). Furthermore, RT-PCR, WB and IHC analysis also show mRNA and tissue level upregulation of α-enolase in OSFWD validating α-enolase as precancer marker.ConclusionsThis study for the first time identifies and validates α-enolase as a novel biomarker for early diagnosis of malignant potentiality of OSF. Hence, the identified protein marker, α-enolase can help in early therapeutic intervention of OSF patients leading to the reduction of patient’s pain, treatment cost and enhancement of patient’s quality of life.

scholarly journals Protein Identification Strategies for the Greenshell Mussel Perna canaliculus

2021 ◽  
Author(s):  
◽  
Cassidy Moeke
Keyword(s):  
Heavy Metal ◽  
Metal Pollution ◽  
Heavy Metal Pollution ◽  
Protein Identification ◽  
De Novo ◽  
De Novo Sequencing ◽  
Cytoskeletal Proteins ◽  
Est Database ◽  
Perna Canaliculus ◽  
Sequence Databases

<p>The greenshell mussel Perna canaliculus is considered to be a suitable biomonitor for heavy metal pollution. This is due to their ability to accumulate and tolerate heavy metals in their tissues. These characteristics make them useful for identifying protein biomarkers of heavy metal pollution, as well as proteins associated with heavy metal detoxification and homeostasis. However, the identification of such proteins is restricted by the greenshell mussel being poorly represented in sequence databases. Several strategies have previously been used to identify proteins in unsequenced species, but only one of these strategies has been applied to the greenshell mussel. The objective of this thesis was to examine different protein identification strategies using a combined two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry approach. The protein identification strategies used include a Mascot database search, as well as de novo sequencing approaches using PEAKS DB and SPIDER homology searches. In total, 155 protein spots were excised and a total of 68 identified. Fifty-six proteins were identified using a Mascot search against the Mollusca, NCBInr and Invertebrate EST database, with seven single-peptide identifications. De novo sequencing strategies identified additional proteins, with two from a PEAKS DB search and 10 from an error-tolerant SPIDER homology search. The most noticeable protein groups identified were cytoskeletal proteins, stress response proteins and those involved in protein biosynthesis. Actin and tubulin made up the bulk of the identifications, accounting for 39% of all proteins identified. This multifaceted approach was shown to be useful for identifying proteins in the greenshell mussel Perna canaliculus. Mascot and PEAKS DB performed equally well, while the error-tolerant functionality of SPIDER was useful for identifying additional proteins. A subsequent search against the Invertebrate EST database was also found to be useful for identifying additional proteins. Despite this, more than half of all proteins remained unidentified. Most of these proteins either failed to produce good quality MS spectra or did not find a match to a sequence in the database. Future research should first focus on obtaining quality MS spectra for all proteins concerned and then examine other strategies that may be more suitable for identifying proteins for species with poor representation in sequence databases.</p>

scholarly journals Vibratory Reaction Unit for the Rapid Analysis of Proteins and Glycochains

2007 ◽  
Vol 2 ◽  
pp. 117739010700200 ◽  
Author(s):  
Yukie Sasakura ◽  
Makoto Nogami ◽  
Noriko Kobayashi ◽  
Katsuhiro Kanda
Keyword(s):  
Mass Spectrometry ◽  
Solid Surface ◽  
Protein Identification ◽  
Immobilized Enzymes ◽  
Rapid Analysis ◽  
Single Step ◽  
Protein Digestion ◽  
Sequence Coverage ◽  
Micro Scale ◽  
Reaction Unit

A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS) analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF), an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG). Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF) would be effective for the rapid structural analysis of glycoproteins.

A Secreted Disulfide Catalyst Controls Extracellular Matrix Composition and Function

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 74-76 ◽  
Author(s):  
Tal Ilani ◽  
Assaf Alon ◽  
Iris Grossman ◽  
Ben Horowitz ◽  
Elena Kartvelishvily ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Disulfide Bond ◽  
De Novo ◽  
Bond Formation ◽  
Supporting Cell ◽  
Matrix Composition ◽  
Secretory Proteins ◽  
Disulfide Bond Formation ◽  
Enzyme Families ◽  
And Function

Disulfide bond formation in secretory proteins occurs primarily in the endoplasmic reticulum (ER), where multiple enzyme families catalyze cysteine cross-linking. Quiescin sulfhydryl oxidase 1 (QSOX1) is an atypical disulfide catalyst, localized to the Golgi apparatus or secreted from cells. We examined the physiological function for extracellular catalysis of de novo disulfide bond formation by QSOX1. QSOX1 activity was required for incorporation of laminin into the extracellular matrix (ECM) synthesized by fibroblasts, and ECM produced without QSOX1 was defective in supporting cell-matrix adhesion. We developed an inhibitory monoclonal antibody against QSOX1 that could modulate ECM properties and undermine cell migration.

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