Hanne Kolsrud Hustoft

Bio: Hanne Kolsrud Hustoft is an academic researcher from University of Oslo. The author has contributed to research in topics: Protein digestion & Proteomics. The author has an hindex of 9, co-authored 9 publications receiving 342 citations. Previous affiliations of Hanne Kolsrud Hustoft include Norwegian University of Life Sciences.

A Critical Review of Trypsin Digestion for LC-MS Based Proteomics

Abstract: Proteomics is defined as the large-scale study of proteins in particular for their structures and functions (Anderson and Anderson 1998), and investigations of proteins have become very important since they are the main components of the physiological metabolic pathways in eukaryotic cells. Proteomics increasingly plays an important role in areas like protein interaction studies, biomarker discovery, cancer prevention, drug treatment and disease screening medical diagnostics (Capelo et al. 2009). Proteomics can be performed either in a comprehensive or “shotgun” mode, where proteins are identified in complex mixtures, or as “targeted proteomics” where “selective reaction monitoring” (SRM) is used to choose in advance the proteins to observe, and then measuring them accurately, by optimizing the sample preparation as well as the LC-MS method in accordance to the specific proteins (Mitchell 2010). Whether “MS-based shotgun proteomics” has accomplished anything at all regarding clinically useful results was recently addressed by Peter Mitchell in a feature article (Mitchell 2010), and he states that the field needs to make a further step or even change direction. Referring to discussions with among others John Yates and Matthias Mann, Mitchell addresses the failure in the search for biomarkers as indicators of disease, the difficulties of protein arrays, the uncertainty of quantification in “shotgun proteomics” (due to among others the efficiency of ionization in the mass spectrometers), database shortcomings, the problems of detecting post translational modifications (PTMs), and finally the huge disappointment in the area of drug discovery. The field points in the direction of targeted proteomics, but targeted proteomics will not be the solution to all our questions and comprehensive proteomics will still be needed. In order to get as much information, with as high quality as possible, from a biological sample, both the sample preparation and the final LC-MS analyses need to be optimized. The most important step in the sample preparation for proteomics is the conversion of proteins to peptides and in most cases trypsin is used as enzyme. Trypsin is a protease that specifically cleaves the proteins creating peptides both in the preferred mass range for MS sequencing and with a basic residue at the carboxyl terminus of the peptide, producing information-rich, easily interpretable peptide fragmentation mass spectra. Some other proteases can be used as well, such as Lys-C, which is active in more harsh conditions with 8 M urea, and give larger fragments than trypsin. Asp-N and Glu-C are also highly sequence-

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

Abstract: A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/s-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.

Shotgun mass mapping of Lactobacillus species and subspecies from caries related isolates by MALDI-MS.

Abstract: A taxonomical study of 90 isolates of lactobacilli isolated from soft and hard carious dentine of 70 deciduous molars is presented. The Lactobacillus strains were determined by shotgun mass mapping (SMM). This method based on MALDI-MS analysis of Lactobacillus isolates treated with trypsin followed by database comparison against a library of mass spectra derived from 20 reference strains. The SMM method allowed to discriminate different Lactobacillus subspecies. The method was used to analyse Lactobacillus isolates of unknown identity derived from carious dentine. Application of the SMM method to isolates from hard carious dentine revealed a nearly similar distribution of L. paracasei ss paracasei (29%), L. paracasei ss tolerans (32%) and L. casei ss rhamnosus (23%) as dominant subspecies. On the other hand, samples derived from soft carious dentine showed a clear bias only to L. paracasei ss paracasei (60%), whereas L. paracasei ss tolerans (14%) and L. casei ss rhamnosus (12%) were clear minorities. Compared to existent methods, SMM has unique potential for the analysis of Lactobacillus strains on subspecies level.

Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5

Abstract: Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.

Electrochemically mediated atom transfer radical polymerization

Abstract: Electrochemical reduction of an exemplary ATRP catalyst, C 11 Br 2 /Me 6 TREN, is shown to be an efficient process to mediate and execute an ATRP. The onset of polymerization occurs only through passage of a cathodic current achieved under a reductive potential to form Cu 1 Br 2 /Me 6 TREN, within the reaction medium. Unprecedented control over the polymerization kinetics can be attained through electrochemical methods by modulating the magnitude of the applied potential allowing polymerization rate enhancement or retardation. Additional polymerization control is gained through electrochemical “dials” allowing polymerization rate enhancements achieved by larger applied potentials and the ability to successfully switch a polymerization “on” and “off between dormant and active states by application of multistep intermittent potentials.

Mass spectrometry tools for the classification and identification of bacteria

Abstract: Mass spectrometry has become an important analytical tool in biology in the past two decades. In principle, mass spectrometry offers high-throughput, sensitive and specific analysis for many applications in microbiology, including clinical diagnostics and environmental research. Recently, several mass spectrometry methods for the classification and identification of bacteria and other microorganisms, as well as new software analysis tools, have been developed. In this Review we discuss the application range of these mass spectrometry procedures and their potential for successful transfer into microbiology laboratories.

MALDI TOF MS profiling of bacteria at the strain level: A review

Abstract: Since the advent of the use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) as a tool for microbial characterization, efforts to increase the taxonomic resolution of the approach have been made. The rapidity and efficacy of the approach have suggested applications in counter-bioterrorism, prevention of food contamination, and monitoring the spread of antibiotic-resistant bacteria. Strain-level resolution has been reported with diverse bacteria, using library-based and bioinformatics-enabled approaches. Three types of characterization at the strain level have been reported: strain categorization, strain differentiation, and strain identification. Efforts to enhance the library-based approach have involved sample pre-treatment and data reduction strategies. Bioinformatics approaches have leveraged the ever-increasing amount of publicly available genomic and proteomic data to attain strain-level characterization. Bioinformatics-enabled strategies have facilitated strain characterization via intact biomarker identification, bottom-up, and top-down approaches. Rigorous quantitative and advanced statistical analyses have fostered success at the strain level with both approaches. Library-based approaches can be limited by effects of sample preparation and culture conditions on reproducibility, whereas bioinformatics-enabled approaches are typically limited to bacteria, for which genetic and/or proteomic data are available. Biological molecules other than proteins produced in strain-specific manners, including lipids and lipopeptides, might represent other avenues by which strain-level resolution might be attained. Immunological and lectin-based chemistries have shown promise to enhance sensitivity and specificity. Whereas the limits of the taxonomic resolution of MALDI TOF MS profiling of bacteria appears bacterium-specific, recent data suggest that these limits might not yet have been reached. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:188–217, 2013

Proteomics for routine identification of microorganisms.

Abstract: The invention of MALDI-TOF-MS enormously contributed to the understanding of protein chemistry and cell biology. Without this technique proteomics would most likely not be the important discipline it is today. Besides 'true' proteomics, MALDI-TOF-MS was applied for the analysis of microorganisms for their taxonomic characterization from its beginning. This approach has since been developed as a diagnostic tool readily available for routine, high-throughput analysis of microbial isolates from clinical specimens by intact-cell mass spectrometry (ICMS), the direct analysis of whole bacterial cell without a preceding fractionation or separation by chromatography or electrophoresis. ICMS exploits the reproducibility of mass fingerprints for individual bacterial and fungal strains as well as the high similarity of mass fingerprints within a species. Comparison of mass spectral data to genomic sequences emphasized the validity of peak patterns as taxonomic markers. Supported by comprehensive databases, MALDI-TOF-MS-based identification has been widely accepted in clinical laboratories within only a few years.