Challenges and developments in protein identification using mass spectrometry

Szabó, Z. G.; Janáky, Tamás

doi:10.1016/j.trac.2015.03.007

Cited by 24 publications

(13 citation statements)

References 91 publications

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“…Protein identification is greatly dependent on bioinformatics (algorithms and protein-sequence databases) (Szabo & Janaky, 2015). A considerable amount of MS data obtained from "bottom-up" proteomics is often too large to integrate manually, so-called de novo sequencing.…”

Section: Quantification Approachesmentioning

confidence: 99%

Proteomics of major bovine milk proteins: Novel insights

Deeth

Larsen

2017

International Dairy Journal

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Milk proteomics covers the identification, characterisation and quantification of milk proteins. Its applications vary from the basic composition of milk proteins to their post-translational modifications (PTMs), occurring naturally or via processing and storage. Through the combination of liquid chromatography or two-dimensional gel electrophoresis with advanced mass spectrometry, milk proteomics has revealed PTMs that affect milk protein structural and functional properties. This review discusses detection by proteomics-based methods with special emphasis on natural and process induced PTMs in the major bovine milk proteins. The review covers the importance of milk proteomics in determining PTMs, especially those formed by heat treatment and during storage, and highlights some breakthroughs in PTM studies. Furthermore, aspects and applications of quantitative proteomics on milk proteins and bioinformatics are covered. ___________________________________________________________________________________ 1998). Milk from other species have been included in other reviews (Cunsolo, Muccilli, Saletti & Foti, 2011; El-Salam, 2014) and will not be included here. Overall, bovine milk proteins and related peptides can be classified into four different groups: caseins (α S1-, α S2-, βand κ-caseins), serum proteins [αlactalbumin (α-La), β-lactoglobulin (β-Lg), bovine serum albumin (BSA), immunoglobulins (Igs) and a range of other minor whey proteins], proteose peptones (low molecular weight peptides derived from caseins and well as proteose peptone component 3, called PP3) and membrane [mostly milk fat globule membrane (MFGM)] proteins (Table 1).

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Section: Quantification Approachesmentioning

confidence: 99%

Proteomics of major bovine milk proteins: Novel insights

Deeth

Larsen

2017

International Dairy Journal

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“…Thanks to the combination of advances in instrumentation, fragmentation methods, and analysis strategies, MS has become an indispensable tool for the study of protein expression, protein interactions, and modifications. [1][2][3][4][5][6][7][8][9][10] A typical strategy for obtaining proteomics information from a biological sample consists of the combination of enzymatic digestion (usually by trypsin) followed by (nano)-liquid chromatography electrospray ionization tandem MS of the resulting peptides. One of the fragmentation approaches is the data-dependent MS/MS analysis (DDA),…”

Section: Introductionmentioning

confidence: 99%

Selection of Collision Energies in Proteomics Mass Spectrometry Experiments for Best Peptide Identification: Study of Mascot Score Energy Dependence Reveals Double Optimum

et al. 2018

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Collision energy is a key parameter determining the information content of beam-type collision induced dissociation tandem mass spectrometry (MS/MS) spectra, and its optimal choice largely affects successful peptide and protein identification in MS-based proteomics. For an MS/MS spectrum, quality of peptide match based on sequence database search, often characterized in terms of a single score, is a complex function of spectrum characteristics, and its collision energy dependence has remained largely unexplored. We carried out electrospray ionization-quadrupole-time of flight (ESI-Q-TOF)-MS/MS measurements on 2807 peptides from tryptic digests of HeLa and E. coli at 21 different collision energies. Agglomerative clustering of the resulting Mascot score versus energy curves revealed that only few of them display a single, well-defined maximum; rather, they feature either a broad plateau or two clear peaks. Nonlinear least-squares fitting of one or two Gaussian functions allowed the characteristic energies to be determined. We found that the double peaks and the plateaus in Mascot score can be associated with the different energy dependence of b- and y-type fragment ion intensities. We determined that the energies for optimum Mascot scores follow separate linear trends for the unimodal and bimodal cases with rather large residual variance even after differences in proton mobility are taken into account. This leaves room for experiment optimization and points to the possible influence of further factors beyond m/ z.

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“…For unambiguous PTM localization either peptide derived from fragmentation ideally carries one possible modification site . Although gentle top‐down analysis was shown to preserve PTMs better than bottom‐up , efficiency of backbone fragmentation depends on the protein sequence , structure and the charge state . Thus, fragmentation of intact proteins cannot always be achieved to the desired extent .…”

Section: Introductionmentioning

confidence: 99%

Top‐down and bottom‐up characterization of nitrated birch pollen allergen Bet v 1a with CZE hyphenated to an Orbitrap mass spectrometer

Gusenkov

Stutz

2018

Electrophoresis

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Tyrosine (Tyr) residues of the major pollen allergen of birch Betula verrucosa, Bet v 1a, were nitrated by peroxynitrite. This modification enhances the allergenicity. Modified tyrosines were identified by analyzing intact allergen variants in combination with top‐down and bottom‐up approaches. Therefore, a laboratory‐built sheath‐liquid assisted ESI interface was applied for hyphenation of CE to an Orbitrap mass spectrometer to localize individual nitration sites. The major focus was on identification of primary nitration sites. The top‐down approach unambiguously identified Tyr 5 as the most prominent modification site. Fragments from the allergen core and the C‐terminal part carried up to three potential nitration sites, respectively. Thus, a bottom‐up approach with tryptic digest was used as a complementary strategy which allowed for the unambiguous localization of nitration sites within the respective peptides. Nitration propensity for individual Tyr residues was addressed by comparison of MS signals of nitrated peptides relative to all cognates of homolog primary sequence. Combined data identified surface exposed Tyr 5 and Tyr 66 as major nitration sites followed by less accessible Tyr 158 whereas Tyr 81, 83 and 150 possess a lower nitration tendency and are apparently modified in variants with higher nitration levels.

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Challenges and developments in protein identification using mass spectrometry

Cited by 24 publications

References 91 publications

Proteomics of major bovine milk proteins: Novel insights

Proteomics of major bovine milk proteins: Novel insights

Selection of Collision Energies in Proteomics Mass Spectrometry Experiments for Best Peptide Identification: Study of Mascot Score Energy Dependence Reveals Double Optimum

Top‐down and bottom‐up characterization of nitrated birch pollen allergen Bet v 1a with CZE hyphenated to an Orbitrap mass spectrometer

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