Method for Identification of Threonine Isoforms in Peptides by Ultraviolet Photofragmentation of Cold Ions

Identification of isomeric amino acid residues in peptides and proteins is challenging but often highly desired in proteomics. One of the practically important cases that require isomeric assignments is that associated with single-nucleotide polymorphism substitutions of Met residues by Thr in cancer-related proteins. These genetically encoded substitutions can yet be confused with the chemical modifications, arising from protein alkylation by iodoacetamide, which is commonly used in the standard procedure of sample preparation for proteomic analysis. Similar to the genetically encoded mutations, the alkylation also induces a conversion of methionine residues, but to the iso-threonine form. Recognition of the mutations therefore requires isoform-sensitive detection techniques. Herein, we demonstrate an analytical method for reliable identification of isoforms of threonine residues in tryptic peptides. It is based on ultraviolet photodissociation mass spectrometry of cryogenically cooled ions and a machine-learning algorithm. The measured photodissociation mass spectra exhibit isoform-specific patterns, which are independent of the residues adjacent to threonine or iso-threonine in a peptide sequence. A comprehensive metric-based evaluation demonstrates that, being calibrated with a set of model peptides, the method allows for isomeric identification of threonine residues in peptides of arbitrary sequence.

Interfacial Complexes between a Protein and Lipophilic Ions at an Oil-Water Interface

Hubert Girault, Rune Hartvig, Manuel Alejandro Méndez Agudelo

The interaction between an intact protein and two lipophilic ions at an oil-water interface has been investigated using cyclic voltammetry, impedance based techniques and a newly developed method in which the biphasic oil-water system is analyzed by biphasic electrospray ionization mass spectrometry (BESI-MS), using a dualchannel electrospray emitter. It is found that the protein forms interfacial complexes with the lipophilic ions and that it specifically requires the presence of the oil-water interface to be formed under the experimental conditions. Furthermore, impedance based techniques and BESI-MS with a common ion to polarize the interface indicated that the Galvani potential difference across the oil-water interface significantly influences the interfacial complexation degree. The ability to investigate protein-ligand complexes formed at polarized liquid-liquid interfaces is thus a new analytical method for assessing potential dependent interfacial complexation using a structure elucidating detection principle.

American Chemical Society2010

Middle-down approaches for mass spectrometry-based protein identification and characterization

Kristina Srzentic

Mass spectrometry (MS) has emerged over the last two decades as the analytical technique of choice in systems-level protein studies, known as proteomics. Two are the MS-based approaches generally applied to proteomics: bottom-up (BU), which relies on the proteolytic digestion of proteins into short (~10 amino acids) peptides, and top-down (TD), where proteolysis is omitted, intact proteins are detected and fragmented in gas phase. Both methods present advantages as well as drawbacks. Here, we sought to establish a complete platform to put forward a third MS-based proteomic approach; middle-down (MD). It implies protein digestion as in BU, but aims to generate large peptides which size approaches the one of small intact proteins that are readily analyzed in TD. This novel domain aims to account for the shortcoming of both classical approaches. Until now, the main reasons behind the limited use of MD proteomics have been the lack of easy-to-use cleaving agents capable of producing peptides in the desired 3-15 kDa mass range with high specificity, limitations in MS and allied instrumentation, and the absence of dedicated bioinformatics tools for processing of acquired data. The latter greatly impedes the next milestone in MD proteomics â large-scale analysis. MD can potentially combine the analysis of large portions of proteins carrying set of biologically-relevant modifications â allowing exploring proteins of molecular weight or complexity incompatible with current TD capabilities â with the high-throughput hallmark of BU proteomics. Here, we first in silico evaluated the potential target amino acid residues to produce peptides in the MD mass range within the proteomes of different organisms. This bioinformatics work was followed by an experimental study based on synthetic MD-sized peptides, aimed at determining the optimal MS and tandem MS parameters for large peptide characterization. Next, we pursued two distinct ways of performing MD proteolysis: i) the use of an enzyme and ii) the use of a chemical reagent. We selected the protease Sap9 as a target enzyme for MD, which we fully characterized and successfully applied to the study of a mixture of monoclonal antibodies, where it showed an advantage over traditional BU in terms of reduced introduction of artifacts to the sample, allowing the post-translational modification investigation and unambiguous antibody identification. The chemical cleavage way we addressed via judicious protocol optimization for hydrolysis at the N-terminal side of cysteine with NTCB reagent. We also advanced MD protocols by generation of large (~50 kDa) subunits of monoclonal antibodies through the use of papain and another more specific novel protease, GingisKHAN, combined with new MS signal processing and data analysis capabilities. The developed workflow improved mapping of the connectivity of cysteines involved in inter- and intra-molecular disulfide bridges in antibodies. To summarize, we demonstrated that MD approach to mass spectrometry and proteomics is a powerful, yet underdeveloped, complement to BU and TD. This work has benchmarked MD for targeted protein analysis. In the near future, with advancements of the field, we envision its growing use for large-scale complex proteome analysis.

EPFL2016

Middle-down approaches for mass spectrometry-based protein identification and characterization

Kristina Srzentic

EPFL2016