Publications
Peer-Reviewed Papers & Preprints
Deep Coverage and Extended Sequence Reads Obtained with a Single Archaeal Protease Expedite de novo Protein Sequencing by Mass Spectrometry
Tatiana M. Shamorkina, Laura Pérez Pañeda, Tereza Kadavá, Douwe Schulte, Patrick Pribil, Sibylle Heidelberger, Allison Michele Narlock-Brand, Steven M. Yannone, Joost Snijder, Albert J. R. Heck
DOI: 10.1101/2025.05.26.656138
The ability to sequence proteins without reliance on a genomic template defines a critical frontier in modern proteomics. This approach, known as de novo protein sequencing, is essential for applications such as antibody sequencing, microbiome proteomics, and antigen discovery, which require accurate reconstruction of peptide and protein sequences. While trypsin remains the gold-standard protease in proteomics, its restricted cleavage specificity limits peptide diversity. This constraint is especially problematic in antibody sequencing, where the functionally critical regions often lack canonical tryptic sites. As a result, exclusively trypsin-based approaches yield sparse reads, leading to sequence gaps. Multi-protease and hybrid-fragmentation strategies can improve the sequence coverage, but they add complexity, compromise scalability and reproducibility.
Here, we explore two HyperThermoacidic Archaeal (HTA)-proteases as single-enzyme solutions for de novo antibody sequencing. Each HTA-protease generated about five times more unique peptide reads than trypsin or chymotrypsin, providing high redundancy across all CDRs. Combined with EAciD fragmentation on a ZenoTOF 7600 system, this approach enabled complete, unambiguous antibody sequencing. De novo analysis using PEAKS/DeepNovo and Stitch showed up to fourfold higher alignment scores and reduced the sequence errors within the HTA-generated data. Additionally, the HTA-EAciD approach offers short digestion times, eliminates extensive cleanup, and enables analysis in a single LC-MS/MS run. This streamlined, single-protease approach delivers therefore performance comparable to multi-enzyme workflows, offering a scalable and efficient strategy for de novo protein sequencing across diverse applications.
Toward Real-Time Proteomics: Blood to Biomarker Quantitation in under One Hour
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
DOI: 10.1021/acs.analchem.4c05172
Multistep multihour tryptic proteolysis has limited the utility of bottom-up proteomics for cases that require immediate quantitative information. The power of proteomics to quantify biomarkers of health status cannot practically assist in clinical care if the dynamics of disease outpaces the turnaround of analysis. The recently available hyperthermoacidic archaeal (HTA) 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 (DTSC) liquid chromatography–mass spectrometry (LC–MS) 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. Furthermore, 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. We discuss some of the implications of real-time proteomics including the immediate potential to advance several clinical and research applications.
From volcanoes to the bench: Advantages of novel hyperthermoacidic archaeal proteases for proteomics workflows
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
DOI: https://doi.org/10.1016/j.jprot.2023.104992
Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate their use for bottom-up proteomic experiments. We find that HTA-Proteases have novel cleavage specificities, show no autolysis, function in dilute formic acid, and store at ambient temperature for years. HTA-Proteases function optimally at 70–90 °C and pH of 2–4 with rapid digestion kinetics. The extreme HTA-Protease reaction conditions actively denature sample proteins, obviate the use of chaotropes, are largely independent of reduction and alkylation, and allow for a one-step/five-minute sample preparation protocol without sample manipulation, dilution, or additional cleanup. We find that brief one-step HTA-Protease protocols significantly increase proteome and protein sequence coverage with datasets orthogonal to trypsin. Importantly, HTA-Protease digests markedly increase coverage and identifications for ribonucleoproteins, histones, and mitochondrial membrane proteins as compared to tryptic digests alone. In addition to increased coverage in these classes, HTA-Proteases and simplified one-step protocols are expected to reduce technical variability and advance the fields of clinical and high-throughput proteomics. This work reveals significant utility of heretofore unavailable HTA-Proteases for proteomic workflows. We discuss some of the potential for these remarkable enzymes to empower new proteomics methods, approaches, and biological insights.
Novel hyperthermoacidic archaeal enzymes for removal of thermophilic biofilms from stainless steel
Yurina Nam, Adam Barnebey, Hayoung K Kim, Steven M Yannone, Steve Flint
DOI: https://doi.org/10.1093/jambio/lxad106
Aims
To test the efficacy of novel hot/acid hyperthermoacidic enzyme treatments on the removal of thermophilic spore-forming biofilms from stainless steel surfaces.
Methods and results
The present study measured the efficacy of hyperthermoacidic enzymes (protease, amylase, and endoglucanase) that are optimally active at low pH (≈3.0) and high temperatures (≈80°C) at removing thermophilic bacilli biofilms from stainless steel (SS) surfaces. Plate counts, spore counts, impedance microbiology, as well as epifluorescence microscopy, and scanning electron microscopy (SEM) were used to evaluate the cleaning and sanitation of biofilms grown in a continuous flow biofilm reactor. Previously unavailable hyperthermoacidic amylase, protease, and the combination of amylase and protease were tested on Anoxybacillus flavithermus and Bacillus licheniformis, and endoglucanase was tested on Geobacillus stearothermophilus. In all cases, the heated acidic enzymatic treatments significantly reduced biofilm cells and their sheltering extracellular polymeric substances (EPS).
Conclusions
Hyperthermoacidic enzymes and the associated heated acid conditions are effective at removing biofilms of thermophilic bacteria from SS surfaces that contaminate dairy plants.
Application Notes
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 for
HTA-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.
Conference Posters & Presentations
HUPO World Congress 2025 Posters
Posters
Illuminating the Dark Proteome with Novel Archaeal Proteases
Simion Kreimer [1], Jennifer Van Eyk [1], Natalie Korkola [2], Kristina Jurcic [2], and Albert J. R. Heck [3], and Steven M. Yannone [4]
[1] Cedars-Sinai Medical Center, Los Angeles, CA, USA [2] Bioinformatics Solutions, Inc. Ontario, Canada [3] Netherlands Proteomics Center, Utrecht, the Netherlands [4] Cinder Biological, Inc. San Leandro CA, USA
Abstract:
Over ninety-percent of the current human proteome map is based on tryptic data sets. Inherent characteristics of novel Archaeal proteases generate sequence and protein identifications that are orthogonal and complimentary to tryptic data and reveal significant new segments of the ‘dark proteome’. We show that the recently introduced hyperthermoacidic archaeal proteases (HTA-Proteases) have unique advantages for expanding our maps of the human proteome. We find that HTA-Proteases preferentially cleave at the carboxy terminus of glutamine, leucine, and phenylalanine residues with incredibly rapid kinetics (5-minute digests). In addition, nucleic acid binding proteins and membrane proteins are highly represented in HTA-data relative to tryptic digests. Our studies further reveal that the extreme HTA-Protease reaction conditions at 80C and pH 3 quench endogenous enzymatic activities in samples thereby capturing data that more closely represents the system status at the time of sampling. We show that labile targets like the angiotensin peptide hormones can be directly quantified in blood with brief and minimal sample manipulation while simultaneously quantifying several circulating blood proteins. We present methods and data that are of utility in expanding the maps of the human proteome and reducing sample-handling artifacts in bottom-up proteomic experiments. Finally, the new methods presented here have clear advantages for increasing throughput and reproducibility of bottom-up proteomics workstreams. We present data from peer-reviewed publications and unpublished data to substantiate our hypotheses and assertions.
ASBMB 2025 Posters
Posters
Archaeal Proteases Facilitate One-Enzyme De Novo Protein Sequencing, Novel Biomarker
Discovery, Rapid Clinical Proteomics and Markedly Accelerate Routine Proteomics Analyses
Simion Kreimer [1] , Jennifer Van Eyk [1], Tatiana M. Shamorkina [3], Laura Pérez Pañeda [3], and Albert J. R. Heck [3], and Steven M. Yannone [2]
[1] Cedars-Sinai Medical Center, Los Angeles, CA, USA [2] Cinder Biological, Inc. San Leandro CA, USA [3] Netherlands Proteomics Center, Utrecht, the Netherlands
Abstract:
The development of recombinant DNA technologies for hyperthermoacidic Archaea (HTA) have
led to the isolation and characterization of a suite of novel enzymes that function optimally at
80-90C at pH 2-4. We have applied the first two available recombinant HTA-Proteases
(Krakatoa and Vesuvius) to bottom-up proteomics analyses and identified many potentially
powerful advantages for the field. Due to the extreme denaturing conditions for optimal HTA-
Proteases function, no chaotropes or alkylation are required, and digests can be run in formic
or any common acid while retaining pH 3. HTA-Proteases show dominant cleavage at E, L, and F
with lesser cleavages at other amino acids. HTA-proteases show remarkably rapid digestion
kinetics with complete digestion being achieved in 5 to 30 minutes. HTA-Protease reactions
condense multi-step multi-hour sample preparations into a one-step, one-tube, 30-minute
sample preparation protocol with no manipulation or sample loss. Protein identifications and
sequence contributions from HTA-Protease digests have a marked bias towards membrane and
nucleic acid binding proteins relative to tryptic digests providing novel data sets and non-
overlapping peptide sets orthogonal and complimentary to tryptic approaches. Moreover, HTA-
Protease approaches have been shown to identify both circulating proteins and peptide-
hormones that are not identified with tryptic approaches in a single run of whole blood in
under one-hour with an eight-minute sampling interval. HTA-Proteases show no autolysis and
require approximately two orders of magnitude less enzyme-mass than mesophilic proteases
for complete digestion of the same sample mass. Most recently, de novo sequencing studies
reveal that both available HTA-Proteases each generated about five times more unique IgG
peptide reads than trypsin or chymotrypsin with high redundancy across all CDRs, providing a
rapid single-enzyme solution for de novo antibody sequencing. Within these studies we find
that less than one-percent of peptides show deamidation or oxidation and leucine cleavage is
robust while isoleucine-terminated peptides represent less than 0.1 percent of identified
peptides, allowing a simple means to discriminate between these amino acids. It can
reasonably be expected that the 2-year-old HTA-Protease technologies will continue to reveal
additional advantages to accelerate and empower proteomics in the biomedical, research, and
clinical applications of proteomics.
ASMS 2025 Posters
Posters
Automatable One-Step 5-minute Sample Preparation with Novel Hyperthermoacidic Proteases Generates Novel Protein ID's and Sequence
ThP 391
Maxwell C. MCCABE [1], Gary SUIZDAK [3], Linh Truc HOANG [3], Keira Y. LARSON [1], Anthony J. SAVIOLA [1], Kirk C. HANSEN [1] and Steven M. YANNONE [2]
[1] University of Colorado Denver, Aurora, CO, USA [2] Cinder Biological, Inc., San Leandro, CA, USA [3] The Scripps Research Institute, La Jolla, CA, USA
Hot de novo Antibody Sequencing: one protease, one run, massively redundant reads covering all hypervariable regions
TP 005
Tatiana M. Shamorkina* [1], Tereza Kadavá* [1], Laura Perez Paneda* [1], Sibylle M. Heidelberger [2], Patrick Pribil [2,3], Steven M. Yannone [4], Joost Snijder [1], Albert J.R. Heck [1]
[1] Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, Utrecht 3584 CH, The Netherlands.
[2] Sciex, Macclesfield, UK
[3] Sciex, Canada
[4] Cinder Biological, USA
* Equal contribution
Unleashing Krakatoa: Assessing a Novel Alternative Enzyme for MS-Based Proteomics
WP 399
Elizabeth M Brown [1], Erik Nash [1], Jennifer Roof [1], Michael Krawitzky [2], Hossein Fazelinia [1,3], Lynn A Spruce [1]
[1] Proteomics Core Facility, Children’s Hospital of Philadelphia, Philadelphia, PA
[2] Bruker Scientific LLC, Billerica, MA
[3] Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA
US HUPO 2025 Presentations & Posters
Presentations
Rapid and Complete Biopharmaceutical Sequencing using a Tunable Hyperthermoacidic Archaeal Protease
Oral Presentation OA10.03; Tues Feb25 11:55am-12:15pm
Steven Yannone, CinderBio; Simion Kreimer, Cedars-Sinai Medical Center
Protein biopharmaceuticals are primarily defined by their amino acid sequence. Host cells are expected to translate the recombinant gene with high fidelity but mutations and errors in translation that compromise safety and efficacy can occur. Thus monitoring of complete expressed protein sequence is required throughout the development and production processes. Current sequencing methods use multiple proteases (up to 6) with complementary cleavage site specificity because an individual protease generates peptides that are too short or too large for unambiguous identification by LC-MS, which results in incomplete sequence coverage. Here we use a Hyoperthermoacidic Archaeal (HTA) protease for proteolysis in an acidic buffer at temperatures >80C to generate redundant peptides of varying lengths that completely cover the entire biopharmaceutical sequence. Our initial optimization of reaction conditions tested 20, 30, and 60 minute duration and varying doses of Vesuvius HTA protease (CinderBio) for sequencing the NIST antibody. Each condition was analyzed using 12 minute data dependent and data independent acquisition (DDA and DIA) on an Exploris 480. Nonspecific protease data processing of the DDA data in PEAKS Studio found 100% sequence coverage. Confirmation of these identification by DIA found that 98.6% of the light and 79% of the heavy chain sequences were covered by peptides with clear elution profiles. Importantly, Vesuvius most frequently cleaves at the C-terminus of leucine residues and infrequently at isoleucine residues, providing an opportunity to distinguish between these isobaric residues as part of the analyses. Ongoing optimization studies will test two additional HTA proteases and their combinations over a range of pH, temperature, and duration. Through these efforts we expect to identify a set of parameters which will generate total sequence coverage from a single optimized reaction thus providing a simple, rapid, scalable, and automatable method for routine sequencing of biopharmaceuticals
Posters
From Volcanoes to the Bench: Advantages of Hyperthermoacidic Archaeal Proteases for Proteomics Workflows
Poster 01.40; Tues Feb 25 4:30-6pm
Steven Yannone, CinderBio; Simion Kreimer, Cedars-Sinai Medical Center; Jennifer Van Eyk, Cedars-Sinai Medical Center; Kirk Hansen, Department of Biochemistry and Molecular Genetics, University of Colorado Denver; Anthony Saviola, Department of Biochemistry and Molecular Genetics, University of Colorado Denver; Maxwell McCabe, Department of Biochemistry and Molecular Genetics, University of Colorado Denver
Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs around the world. We find that HTA-Proteases have novel cleavage specificity, show no autolysis, and store in aqueous buffer at ambient temperature for over two years. HTA-Proteases function optimally at 70-90°C and pH of 2-4 with very rapid digestion kinetics. The extreme HTA-Protease reactions thermally and chemically denature sample proteins and block disulfides and thereby obviate the use of chaotropes or alkylation. These new capabilities ultimately allow for a brief digestion of plasma in under one hour. Finally, we have applied HTA-Protease technologies to de-novo sequencing of antibodies and achieved 100% coverage with a single-HTA-enzyme in under one hour.
US HUPO 2024 Posters
Posters
P01.37 | From Volcanoes to the Bench: Advantages of Novel Hyperthermoacidic Archaeal Proteases for Proteomics Workflows
Monday March 11, 13:30-15:00
Steven M. Yannone [1], Maxwell McCabe [2], Varun Gejji [1], Adam Barnebey [1], G. Suizdak [3], Lihn T. Hoang [3], Anthony J Saviola [2], Kirk Hansen [2]
Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs around the world. HTA-Proteases function optimally at 70- 90°C and pH of 2-4 with rapid digestion kinetics. The extreme heat and acidity of HTA-Protease reactions denatures sample proteins and obviates the use of chaotropes and facilitates one-step/five-minute sample preparation protocol without sample manipulation, dilution or cleanup thereby reducing/eliminating sample loss. We also find that HTA-Proteases have novel cleavage specificity, show no autolysis, function in dilute formic acid, and ship and store in aqueous buffer at ambient temperature for over two years. We find that brief one-step HTA-Protease protocols significantly increase proteome and protein sequence coverage with datasets orthogonal to trypsin. Importantly, HTA-Protease digests show a bias for identification of ribonucleoproteins, histones, mitochondrial, and membrane proteins. HTA-Protease one-step protocols in dilute formic acid are of possible advantage for many areas of proteomic research.
[1] CinderBio, San Leandro, CA)
[2] Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Denver, CO
[3] The Scripps Research Institute, La Jolla, CA
P03.08 | Near Real-Time Monitoring of Blood Biomarkers with “Rapid Proteomics”
Tuesday March 12, 13:30-15:00
Steven M. Yannone [1], Jennifer Van Eyk [2], Simion Kreimer [2]
Multi-hour tryptic proteolysis disqualifies conventional bottom-up proteomics from diagnosis of acute clinical conditions because a fast turn-around is needed to monitor and mitigate rapidly progressing physiological reactions. The Krakatoa protease manufactured by CinderBio proteolyzes samples at pH 3 and 80-90 C in just 10-30 minutes. “Rapid Proteomics” leverages quick sample preparation and high throughput LC-MS for a turn-around of 30-60 minutes from blood collection to actionable data to quantify circulating biomarkers in near real-time. The Krakatoa protease most frequently cleaves Leucine, Phenylalanine, and Glutamic acid but with limited specificity and many missed cleavages. Nonetheless qualification of a 20-minute proteolysis of 1 microliter of venous blood from a finger prick with a 20-minute data independent acquisition (DIA) LC-MS analysis found that over 160 circulating proteins were reproducibly quantified. Over 100 proteins were quantified at CV <30% inter-day imprecision based on 5 replicates prepared on 3 different days (n = 15) in blood from 3 healthy donors. Over 120 proteins had a CV <20% intraday imprecision across all 3 days (5 replicates in one batch). Optimization of the proteolysis duration spanning 10 to 60 minutes found that proteins were digested after just 15 minutes while the larger peptide were cleaved after 60 minutes. This indicates that the proteolysis strongly favors large substrates and circulating bioactive peptide hormones like Angiotensin I and II and Bradykinin are retained and protected from endogenous proteases by the acidity of the digestion buffer. An 8-minute targeted LC-MS method was developed to quantify bradykinin and angiotensin variants after a 20-minute proteolysis of 1 microliter of venous blood. This inaugural Rapid Proteomics method allows measurement of these critical hormones and several additional proteins at 8-minute intervals with a 30-minute delay to monitor Phase 1 drug responses, allergic challenge reactions, and other other applications.
[1] CinderBio, San Leandro, CA)
[2] Cedars-Sinai Medical Center, Los Angeles, CA
