Identifying proteins and post-translational modifications by mass spectrometry

doi:10.1016/S0959-440X(98)80075-4

Current Opinion in Structural Biology

Volume 8, Issue 3, June 1998, Pages 393-400

https://doi.org/10.1016/S0959-440X(98)80075-4 Get rights and content

Abstract

Major recent advances in hardware performance, sample-handling procedures and software algorithms now allow reliable and sensitive mass spectrometric identification of proteins. Mass spectrometry vastly outperforms traditional sequencing technologies and thereby greatly facilitates the elucidation of the functions of individual proteins as well as multiprotein complexes and larger protein assemblages.

References (63)

M Mann et al.
Developments in matrix-assisted laser desorption/ionization peptide mass spectrometry
Curr Opin Biotechnol
(1996)
H Morris et al.
High-sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer
Rapid Commun Mass Spectrom
(1996)
ON Jensen et al.
Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching
Anal Chem
(1997)
RR Ogorzalek Loo et al.
Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping
Electrophoresis
(1997)
C Eckerskorn et al.
Analysis of proteins by direct-scanning infrared-MALDI mass spectrometry after 2D-PAGE separation and electroblotting
Anal Chem
(1997)
AL McCormack et al.
Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level
Anal Chem
(1997)
JL Joyal et al.
Calmodulin modulates the interaction between IQGAP1 and Cdc42. Identification of IQGAP1 by nanoelectrospray tandem mass spectrometry
J Biol Chem
(1997)
H Deissler et al.
Rapid protein sequencing by tandem mass spectrometry and cDNA cloning of p20-CGGBP. A novel protein that binds to the unstable triplet repeat 5′-d(CGG)n-3′ in the human FMR1 gene
J Biol Chem
(1997)
T Kawata et al.
SH2 signaling in a lower eukaryote: a STAT protein that regulates stalk cell differentiation in dictyostelium
Cell
(1997)
F Mercurio et al.
IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF- kappaB activation
Science
(1997)

P Mitchell et al.

The exosome: a conserved eukaryotic RNA processing complex containing multiple 3′→5′ exoribonucleases

Cell

(1997)

VC Wasinger et al.

Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium

Electrophoresis

(1995)

A Shevchenko et al.

Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels

Proc Natl Acad Sci USA

(1996)

AJ Link et al.

Identifying the major proteome components of Haemophilus influenzae type-strain NCTC 8143

Electrophoresis

(1997)

M Wilm et al.

Parent ion scans of unseparated peptide mixtures

Anal Chem

(1996)

M Karas et al.

Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons

Anal Chem

(1988)

RS Brown et al.

Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometer

Anal Chem

(1995)

U Bahr et al.

Delayed extraction time-of-flight MALDI mass spectrometry of proteins above 25,000 Da

J Mass Spectrometry

(1997)

RM Whittal et al.

Functional wave time-lag focusing matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer: improved mass accuracy

Anal Chem

(1997)

JB Fenn et al.

Electrospray ionization for mass spectrometry of large biomolecules

Science

(1989)

M Wilm et al.

Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry

Nature

(1996)

M Wilm et al.

Analytical properties of the nanoelectrospray ion source

Anal Chem

(1996)

Q Xue et al.

Multichannel microchip electrospray mass spectrometry

Anal Chem

(1997)

D Figeys et al.

A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry

Anal Chem

(1997)

KR Jonscher et al.

The quadrupole ion trap mass spectrometer — a small solution to a big challenge

Anal Biochem

(1997)

M Guilhaus et al.

Perfect timing: time-of-flight mass spectrometry

Rapid Commun Mass Spectrom

(1997)

A Shevchenko et al.

Rapid ‘de novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer

Rapid Commun Mass Spectrom

(1997)

WJ Henzel et al.

Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases

Proc Natl Acad Sci USA

(1993)

M Mann et al.

Use of mass spectrometric molecular weight information to identify proteins in sequence databases

Biol Mass Spectrom

(1993)

D Pappin et al.

Rapid identification of proteins by peptide mass finger printing

Curr Biol

(1993)

P James et al.

Protein identification by mass profile fingerprinting

Biochem Biophys Res Commun

(1993)

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Femtosecond laser-induced dissociation (fs-LID) as an activation method in mass spectrometry
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Common PTMs are phosphorylation or glycosylation. The modifications are rather labile and are not easily detected [32,28]. For the peptide 1P, we observed sequence ions containing phosphorylated serine (S[p]) with the intact PTM.
We report the photolysis of biomolecules in a Fourier-transform ion cyclotron resonance mass spectrometer by intense near-infrared femtosecond laser pulses. Photo-fragmentation was accompanied by photo-ionization and created a large number and variety of charged fragments, which could be identified with high confidence due to the outstanding resolving power and mass accuracy of the mass spectrometer. Fragmentation patterns were sufficient for peptide sequence analysis. Fragments formed by non-ergodic cleavage retained labile post-translational modifications.
Elucidating structural and molecular mechanisms of β-arrestin-biased agonism at GPCRs via MS-based proteomics
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The discovery of β-arrestin-dependent GPCR signaling has led to an exciting new field in GPCR pharmacology: to develop “biased agonists” that can selectively target a specific downstream signaling pathway that elicits beneficial therapeutic effects without activating other pathways that elicit negative side effects. This new trend in GPCR drug discovery requires us to understand the structural and molecular mechanisms of β-arrestin-biased agonism, which largely remain unclear. We have used cutting-edge mass spectrometry (MS)-based proteomics, combined with systems, chemical and structural biology to study protein function, macromolecular interaction, protein expression and posttranslational modifications in the β-arrestin-dependent GPCR signaling. These high-throughput proteomic studies have provided a systems view of β-arrestin-biased agonism from several perspectives: distinct receptor phosphorylation barcode, multiple receptor conformations, distinct β-arrestin conformations, and ligand-specific signaling. The information obtained from these studies offers new insights into the molecular basis of GPCR regulation by β-arrestin and provides a potential platform for developing novel therapeutic interventions through GPCRs.
Protein species as diagnostic markers
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The authors also state, that the interpretation of DNA sequencing in the presence of inserted nucleotide sequences in the heterozygous state can be difficult and requires direct and reverse sequencing. MS-driven protein analysis can be applied to check and validate DNA sequencing results and detect Hb species (Hb Bristol [252,253] Gluthionated Hb “HbSSG” [126], Fetal Hb F Texas [254]) differing in their posttranslational modifications such as acetylation, deamidation (Providence Asp [255], Redondo, Hb J Sardegna [256]), oxidation products [257,258] or artifacts [259,260]. Kleinert et al. identified two additional Hb variants via MS that were missed using standard protein methods [261].
Many diseases are associated with protein species perturbations. A prominent example of an established diagnostic marker is the glycated protein species of hemoglobin, termed HbA1c. HbA1c concentration is increased in the blood of diabetes mellitus patients due to their poor control of blood glucose levels resulting in an increased non-enzymatic glycosylation of hemoglobin producing HbA1c. This important diagnostic marker is routinely measured in the blood of diabetes patients. As in the case of HbA1c, protein species can mirror pathophysiological events. Shifts in the levels of protein species can be associated with or even be responsible for disease making them well suited as diagnostic markers. However, only a few protein species are currently used as diagnostic markers in routine clinical chemistry laboratories, despite being widely established in clinical proteomics research. This review provides an overview of the biochemical characteristics associated with protein species as well as examples of pathophysiological mechanisms, which cause modifications in the protein species composition, thereby emphasizing the importance of screening for protein markers at the species level. Further, we highlight techniques, which are currently utilized for investigating protein species markers in clinical research.
The success rate of FDA approved diagnostic protein markers until today is very low compared to the number of published candidate disease markers. It is hypothesized that one important reason is the gene-centric view which is still followed in clinical proteomics: In many investigations proteins are still digested in small peptides thus making it nearly impossible to discriminate between healthy proteins and pathologic proteins causing diseases. Thus this review is focusing on the biochemistry and patho-biochemistry of proteins, is highlighting the need for screening for disease markers on the protein species level and is giving an overview about available techniques.
Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy
2011, Journal of Biological Chemistry
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In any case, detection of this cleavage event provides clear evidence for donor site identification. Although MS is widely used in the analysis of post-translational protein modifications (44–46), it has rarely been used to identify iodinated tyrosyl residues in Tg and only on fragmented Tg (3, 6, 17). Here, we report the first detailed analysis of the entire Tg molecule from a direct, fresh extract of the mouse thyroid gland.
Thyroglobulin (Tg) is secreted by thyroid epithelial cells. It is essential for thyroid hormonogenesis and iodine storage. Although studied for many years, only indirect and partial surveys of its post-translational modifications were reported. Here, we present a direct proteomic approach, used to study the degree of iodination of mouse Tg without any preliminary purification. A comprehensive coverage of Tg was obtained using a combination of different proteases, MS/MS fragmentation procedures with inclusion lists and a hybrid mass high-resolution LTQ-Orbitrap XL mass spectrometer. Although only 16 iodinated sites are currently known for human Tg, we uncovered 37 iodinated tyrosine residues, most of them being mono- or diiodinated. We report the specific isotopic pattern of thyroxine modification, not recognized as a normal peptide pattern. Four hormonogenic sites were detected. Two donor sites were identified through the detection of a pyruvic acid residue in place of the initial tyrosine. Evidence for polypeptide cleavages sites due to the action of cathepsins and dipeptidyl proteases in the thyroid were also detected. This work shows that semi-quantitation of Tg iodination states is feasible for human biopsies and should be of significant medical interest for further characterization of human thyroid pathologies.
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Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RPLC–MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC–MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC–MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU–PAGE separation and nano-LC–MS/MS.
Identification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass spectrometry
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Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein profiling is described in which mammalian cells are lysed directly in the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) and mass analyzed using MALDI-time of flight (TOF). Using the unique MALDI mass spectral “fingerprint” generated in these analyses, it is possible to differentiate among several different mammalian cell lines. A number of techniques, including MALDI-post source decay (PSD), MALDI tandem time-of-flight (TOF-TOF), MALDI-Fourier transform ion cyclotron resonance (FTICR), and nanoflow liquid chromatography followed by electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) were employed to attempt to identify the proteins represented in the MALDI spectra. Performing a tryptic digestion of the supernatant of the cells lysed in DHB with subsequent LC-ESI-MS/MS analysis was by far the most successful method to identify proteins.

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Identifying proteins and post-translational modifications by mass spectrometry

Abstract

Curr Opin Biotechnol

Rapid Commun Mass Spectrom

Anal Chem

Electrophoresis

Anal Chem

Anal Chem

J Biol Chem

J Biol Chem

Cell

Science

Cell

Electrophoresis

Proc Natl Acad Sci USA

Electrophoresis

Anal Chem

Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons

Anal Chem

Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometer

Anal Chem

Delayed extraction time-of-flight MALDI mass spectrometry of proteins above 25,000 Da

J Mass Spectrometry

Functional wave time-lag focusing matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer: improved mass accuracy

Anal Chem

Electrospray ionization for mass spectrometry of large biomolecules

Science

Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry

Nature

Analytical properties of the nanoelectrospray ion source

Anal Chem

Multichannel microchip electrospray mass spectrometry

Anal Chem

A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry

Anal Chem

The quadrupole ion trap mass spectrometer — a small solution to a big challenge

Anal Biochem

Perfect timing: time-of-flight mass spectrometry

Rapid Commun Mass Spectrom

Rapid ‘de novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer

Rapid Commun Mass Spectrom

Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases

Proc Natl Acad Sci USA

Use of mass spectrometric molecular weight information to identify proteins in sequence databases

Biol Mass Spectrom

Rapid identification of proteins by peptide mass finger printing

Curr Biol

Protein identification by mass profile fingerprinting

Biochem Biophys Res Commun