Tandem Mass Spectrometry of Ribonuclease A and B:  N-Linked Glycosylation Site Analysis of Whole Protein Ions

Reid, Gavin E.; Stephenson, James L.; McLuckey, Scott A.

doi:10.1021/ac015618l

Cited by 75 publications

(100 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…MS/MS by CID has proved to be a successful approach in the identification of histone PTMs, such as glycosylation [130], phosphorylation [131], acylation [132], acetylation [87,133] and methylation [87,134]. FIGURE 4 illustrates one example of such application.…”

Section: Tandem Msmentioning

confidence: 99%

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Ren

Freitas

2007

Expert Review of Proteomics

View full text Add to dashboard Cite

Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications. Keywordshistone; mass spectrometry; post-translational modification Histones are a group of highly conserved proteins throughout eukaryotic evolution [1]. The core histones (H4, H3, H2A and H2B) form an octamer, in which a H2A-H2B dimer associates on each side of the H3-H4 tetramer through an interaction between the C-terminal halves of H2B and H4 [2]. The negatively charged DNA superhelix wraps around the histone octamer to form repeating nucleosome cores, which further assemble into higher order chromatin fibers stabilized by the linker histones (H1 and H5) and linker DNA [3]. The histone fold regions in core histones are highly conserved α-helices, which are connected by two loops. The third histone structural motif comprises the flexible and irregular histone tails, which are extended and reach outside of the nucleosomal unit to interact with DNA [4]. Challenge of molecular characterization: post-translational modifications & sequence variationsAs a building block of the nucleosome, histones play an important role in chromatin assembly and affect the interactions between DNA and other chromatin-associated proteins. Histones regulate gene transcription through their diverse post-translational modifications (PTMs), which include lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine ubiquitination, lysine sumoylation, and poly-ADP-ribosylation of glutamic acid [5][6][7][8]. Among all the PTMs, histone acetylation and methylation have been most †

show abstract

Section: Tandem Msmentioning

confidence: 99%

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Ren

Freitas

2007

Expert Review of Proteomics

View full text Add to dashboard Cite

show abstract

“…The different glycoform was clearly identified. The peak at m/z 13,688 was most likely caused by the loss of the entire glycan by°in-source°decay° [3].°Fibrinogen°is°a°high°molecular weight blood glycoprotein. It usually needs high PBS buffer concentration for solvation because of the high molecular°weight°and°the°attached°glycan° [14].°There-fore, the ionization of a whole protein is usually difficult.…”

Section: Protein Immobilization On Apba Magnetic Beadsmentioning

confidence: 99%

“…The large discrepancy between the extreme diversity of the glycoforms found in nature and thus, the development of a sensitive and specific technique for their elucidation, was required. Mass spectrometry has been proven to be particularly useful for the analysis of protein glycosylation [3][4][5]. Glycosylation site analysis is usually performed following proteolytic cleavage of the glycoprotein, such that each glycosylation site is located within a separate peptide.…”

mentioning

confidence: 99%

Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry

Lee¹,

Kim²,

et al. 2005

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Aminophenylboronic acid (APBA) has been immobilized on magnetic beads for the direct determination of glycoprotein by matrix assisted laser desorption/ionizaton time of flight mass spectrometry (MALDI-TOF-MS). An APBA layer was formed on the surface of carboxylic acid terminated magnetic beads by coupling with carbodiimide and subsequently reacted with an N-hydroxysuccinimide moiety. The immobilized APBA was identified by MALDI-TOF-MS without a matrix. Glycoproteins, such as HbA1c, fibrinogen, or RNase B were separated and desalted using APBA magnetic beads by simply washing the magnetic beads and then separating them by external magnet. Proteins can be identified by direct determination of proteins on beads on MALDI plate and confirmed again by peptide mass finger printing after digestion of proteins on magnetic beads by trypsin. Fluorescence image with a FITC tagging protein using confocal laser microscopy showed the difference of immobilization efficiency between glycoproteins and nonglycoproteins. The methods developed within this work allow the simple treatment and enrichment of glycoproteins as well as direct determination of proteins on beads by

show abstract

“…The fragmentation of a variety of proteins with different charge states was extensively investigated in McLuckey's laboratory, including human hemoglobin ␣-and ␤-chain ions [22,23], insulin [24], apomyoglobin [25], ubiquitin [24], ferro-, ferri-, and apo-cytochrome c ion [26], ribonuclease A and B [27], and native and reduced elastase [6]. To summarize this body of work, more primary structure information is obtained when intermediate charge states are subjected to CID and post ion/ion reaction.…”

mentioning

confidence: 99%

Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry

Liu

Schey

2008

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach. ragmentation of intact protein gas-phase ions, termed top down proteomics, is becoming more frequently utilized primarily in ion trapping instruments. As was first reported by Loo et al.[1], multiply-charged intact protein ions are formed via electrospray ionization (ESI) and can be collisionally activated to produce sequence-specific fragment ions. Typically, the fragmentation of highly multiply charged intact proteins results in a product ion spectrum displaying a multitude of product ions possessing a range of charge states making spectral interpretation challenging. The high resolving power and mass accuracy of Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry has made this instrument platform the method of choice for the interpretation of the complicated product ion spectra produced by fragmentation of multiply charged protein ions. Indeed, automated interpretation algorithms have been constructed based on this approach [2,3]. Using FTMS, proteins over 200 kDa have been successfully fragmented and sequence-specific product ion assignments have been made [4]. Lower mass resolving quadrupole ion trapping instruments have also been successfully utilized for top down proteomics experiments. To meet the challenge of spectral interpretation ion/ion reactions have been employed to reduce the product ion charge by subjecting the product ion population to proton transfer reactions, thereby resulting in the formation of predominantly singly charged product ions and a more readily interpretable product ion spectrum [5]. This strategy removes the product ion charge-state ambiguities arising from dissociation of large multiply charged protein ions and has been successfully demonstrated on ions as large as elastase (25.9 kDa) although it is limited by the m/z range of the instrument [6]. Matrix-assisted laser desorption ionization tandem time-of-flight (MALDI TOF-TOF) mass spectrometry has also been used to produce product i...

show abstract

Tandem Mass Spectrometry of Ribonuclease A and B: N-Linked Glycosylation Site Analysis of Whole Protein Ions

Cited by 75 publications

References 45 publications

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry

Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry

Contact Info

Product

Resources

About