Publications
Laura Perez Paneda, Tereza Kadava, Tatiana M. Shamorkina, Douwe Schulte, Patrick Pribil, Sibylle Heidelberger, Allison Michele Narlock-Brand, Steven M. Yannone, Joost Snijder, and Albert J.R. Heck
AUTHORS
JOURNAL
Deep coverage and extended sequence reads obtained with a single archaeal protease expedite de novo protein sequencing by mass spectrometry
High Throughput Proteomics
Antibody Sequencing
March 11, 2026
The ability to sequence proteins without reliance on a genomic template defines a critical frontier in proteomics. This approach, known as de novo protein sequencing, is essential for applications in antibody sequencing, microbiome proteomics, and antigen discovery, which require accurate reconstruction of target sequences. To advance this field, we explore two hyperthermoacidic archaeal proteases for de novo antibody sequencing, benchmarking them against trypsin and chymotrypsin. Each HTA-protease generated about five times more unique peptide reads than trypsin or chymotrypsin, providing high redundancy across all complementarity-determining regions. Combined with EAciD fragmentation on a ZenoTOF, this methodology enabled complete, unambiguous antibody sequencing. De novo analysis showed much higher alignment scores and reduced the sequence errors by using the HTA-generated data.
ABSTRACT
Steven M. Yannone, Vikas Tuteja, Olena Goleva, Donald Y. M. Leung, Aleksandr Stotland, Angel J. Keoseyan, Nathan G. Hendricks, Sarah Parker, Jennifer E. Van Eyk, and Simion Kreimer
AUTHORS
JOURNAL
Toward Real-Time Proteomics: Blood to Biomarker Quantitation in under One Hour
High Throughput Proteomics
Difficult Proteins
March 20, 2025
Hyperthermoacidic archaeal protease “Krakatoa” digests samples in a single 5 to 30 min step at pH 3 and >80 °C in conditions that disrupt most cells and tissues, denature proteins, and block disulfide reformation thereby dramatically expediting and simplifying sample preparation. The combination of quick single-step proteolysis with high-throughput dual-trapping single analytical column liquid chromatography–mass spectrometry returns actionable data in less than 1 h from collection of unprocessed biofluid. The systematic evaluation of this methodology finds that over 160 proteins are quantified in less than 1 h from 1 μL of whole blood. Labile Angiotensin I and II bioactive peptides along with a panel of protein species can be measured at 8 min intervals with a 20 min initial lag using targeted MS. With these methods, we analyzed serum and plasma from 53 individuals and quantified Angiotensin I and II and over 150 proteins including at least 46 that were not detected with trypsin.
ABSTRACT
Biomarker Discovery
Maxwell C. McCabe, Varun Gejji,, Adam Barnebey , Gary Siuzdak , Linh Truc Hoang , Truc Pham , Keira Y. Larson , Anthony J. Saviola, Steven M. Yannone, Kirk C. Hansen
AUTHORS
JOURNAL
From volcanoes to the bench: Advantages of novel hyperthermoacidic archaeal proteases for proteomics workflows
How To Use
Difficult Proteins
October 30, 2023
HTA-Proteases have novel cleavage specificity, require no chaotropes, and allow simple one-step/five-minute sample preparations that promise to reduce variability between samples and laboratories. They generate unique sets of observable peptides that are non-overlapping with tryptic peptides and significantly increase sequence coverage and available peptide targets relative to trypsin alone. HTA-Proteases show some bias for the detection and coverage of nucleic acid-binding proteins and membrane proteins relative to trypsin. These new ultra-stable enzymes function optimally in nearly boiling acidic conditions, show no autolysis, and do not require aliquoting as they are stable for years at ambient temperatures. Used independently or in conjunction with tryptic digests, HTA-Proteases offer increased proteome coverage, unique peptide targets, brief one-step protocols amenable to automation, rapid turnaround, and high-throughput approaches.
ABSTRACT
Yurina Nam, Adam Barnebey, Hayoung K Kim, Steven M Yannone, Steve Flint
AUTHORS
JOURNAL
Novel hyperthermoacidic archaeal enzymes for removal of thermophilic biofilms from stainless steel
Industrial
May 22, 2023
Hyperthermoacidic archaeal enzymes function optimally in conditions that are toxic to most microbes and are tested here for cleaning and removal of biofilms. Anoxybacillus flavithermus, B. licheniformis, and G. stearothermophilus are abundant thermophilic biofilm-formers in the dairy industry. Due to the inadequate hygienic performance of conventional cleaning approaches on biofilms, enzymes have been studied as an alternative, predominately on non-spore-forming mesophilic or psychrophilic biofilms at a basic or neutral pH at ≈60°C. This study showed the potential for HTA-enzymes as natural and effective cleaning and sanitation products for the removal of biofilms. Additionally, the use of enzymatic cleaning formulations will reduce the environmental impacts caused by the disposal of traditional cleaners such as NaOH and quaternary ammonium compounds (QACs).
ABSTRACT
Lauren G. Poole, Lauren R. Schmitt, Anthony Schulte, Dafna J. Groeneveld, Holly M. Cline, Yaqiu Sang, Woosuk S. Hur, Alisa S. Wolberg, Matthew J. Flick, Kirk C. Hansen, James P. Luyendyk
AUTHORS
JOURNAL
Altered fibrinogen γ-chain cross-linking in mutant fibrinogen-γ mice drives acute liver injury
April 3, 2023
Acetaminophen (APAP)-induced liver injury involves hepatic accumulation of cross-linked fibrin(ogen). To define the role of the fibrinogen γ-chain C-terminal integrin αIIbβ3 binding domain, researchers challenged wild-type and FibγΔ5 mice (which express mutant fibrinogen unable to bind αIIbβ3) with APAP. Following the challenge, FibγΔ5 mice showed altered hepatic fibrin(ogen) deposition and abnormal γ-chain cross-linking. Notably, they lacked the γ-γ dimer and accumulated larger complexes. Mass spectrometry revealed this defect originates from the absence of a critical lysine residue in the mutant protein. The accumulation of this uniquely aberrant, cross-linked fibrin(ogen) exacerbated liver injury in FibγΔ5 mice, an effect not observed by just inhibiting integrin αIIbβ3. The study concludes that fibrinogen-γΔ5 lacks the residues required to form normal γ-γ dimers, and this abnormal cross-linking directly worsens APAP-induced hepatic damage.
ABSTRACT
Δ5
Difficult Proteins
Biomarker Discovery
Sean Reichard
AUTHOR
SOURCE
CinderBio Derives New Enzyme From Yellowstone Hot Springs
November 11, 2015
Industrial
Berkeley startup CinderBio has engineered highly heat- and acid-resistant enzymes derived from Yellowstone National Park's extreme bacteria. These unique enzymes can replace harsh chemicals in industrial processes like food equipment cleaning. In successful creamery trials, they reduced water use by 30% while cleaning faster and more effectively.
Application Notes
How To Use
Determination of unknown PTMs and increased protein sequence coverage from high temperature acidic enzyme digests
Natalie Korkola, Bioinformatics Solutions Inc., Waterloo, Canada
It is important to consider the appropriate PTMs in proteomics searches, to obtain the most complete data possible. However, it is not always known which PTMs to select in advance of a search. In particular, atypical digestion conditions such as the high temperatures and low pH used for hyperthermoacidic archaeal (HTA) enzyme digestions may produce different PTMs during sample preparation than those produced during a typical tryptic digest. Here, the PEAKS PTM algorithm, part of the PEAKS Studio 13 software package, is used to determine the differences in PTMs between a tryptic digest and HTA digests. This approach yielded an 8% increase in protein ID’s forHTA-protease data and boosted the number of protein ID’s seen only with HTA digests from 91 to 248 proteins.
Full de novo sequence coverage and complete automatic assembly of an IgG using a single hyperthermoacidic archaeal enzyme and PEAKS AB 3.5
Natalie Korkola, Bioinformatics Solutions Inc. Waterloo, Canada
Antibody sequencing usually requires performing multiple time-consuming, orthogonal enzyme digests for adequate sequence coverage and assembly. In this application note, complete de novo sequencing coverage and automatic assembly of human IgG is achieved using a single enzyme digest and PEAKS AB 3.5. The novel use of a single hyperthermoacidic archaea enzyme for complete, accurate sequence coverage is unprecedented.
Antibody Sequencing
Conference Posters & Presentations
ASMS 2025 Posters
Automatable One-Step 5-minute Sample Preparation with Novel Hyperthermoacidic Proteases Generates Novel Protein ID's and Sequence
Biomarker Discovery
Difficult Proteins
Antibody Sequencing
Hot de novo Antibody Sequencing: one protease, one run, massively redundant reads covering all hypervariable regions
How To Use
Unleashing Krakatoa: Assessing a Novel Alternative Enzyme for MS-Based Proteomics
US HUPO 2025 Presentations & Posters
Biomarker Discovery
Presentation
Rapid and Complete Biopharmaceutical Sequencing using a Tunable Hyperthermoacidic Archaeal Protease
Difficult Proteins
How To Use
High Throughput Proteomics
Difficult Proteins
Poster
From Volcanoes to the Bench: Advantages of Hyperthermoacidic Archaeal Proteases for Proteomics Workflows