Collision-induced reporter fragmentations for identification of covalently modified peptides

Hung, Chien‐Wen; Schlösser, Andreas; Lehmann, Wolf D.

doi:10.1007/s00216-007-1449-y

Cited by 37 publications

(40 citation statements)

References 79 publications

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“…Surprisingly, there are almost as many b-ions as there are y-ions. As expected [19], the b 1 ion is not observed and the b 2 ion is the most frequent one. After this the distribution of b-ions decreases until b 4 , where it stays about constant until it catches up to the number of y-ions at b 11 /y 11 .…”

Section: Properties Of Identifiable Tandem Mass Spectrasupporting

confidence: 83%

How much peptide sequence information is contained in ion trap tandem mass spectra?

Cox

Hubner

Mann

2008

J. Am. Soc. Mass Spectrom.

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Matching peptide tandem mass spectra to their cognate amino acid sequences in databases is a key step in proteomics. It is usually performed by assigning a score to a spectrum-sequence combination. De novo sequencing or partial de novo sequencing is useful for organisms without sequenced genome or for peptides with unexpected modifications. Here we use a very large, high accuracy proteomic dataset to investigate how much peptide sequence information is present in tandem mass spectra generated in a linear ion trap (LTQ). More than 400,000 identified tandem mass spectra from a single human cancer cell line project were assigned to 26,896 distinct peptide sequences. The average absolute fragment mass accuracy is 0.102 Da. There are on average about four complementary b-and y-ions; both series are equally represented but y ions are 2-to 3-fold more intense up to mass 1000. Half of all spectra contain uninterrupted b-or y-ion series of at least six amino acids and combining b-and y-ion information yields on average seven amino acid sequences. These sequences are almost always unique in the human proteome, even without using any precursor or peptide sequence tag information. Thus, optimal de novo sequencing algorithms should be able to obtain substantial sequence information in at least half of all cases. , and many others score these MS/MS spectra against in silico digested peptides whose calculated precursor masses fall into a suitable window around the measured mass, leading to statistically significant identification for a fraction of the mass spectrometric sequencing events [5]. In most cases, the proportion of identifiable peptides is quite low for samples of high protein complexity [6]. Despite recent improvements in identification rates [7,8], many MS/MS spectra remain unassigned, even though they are of reasonable quality.The peptide database search approach has the disadvantage that it is blind towards the unexpected: only peptides that result from the digestion of known protein sequences, possibly having a few missed cleavages and a very limited number of standard variable modifications, can be identified in this way. The sequence tag approach [9] is an alternative to the conventional peptide database search that does not suffer from these limitations. Instead of operating in the restricted space of in silico digestions of known protein sequences, one starts by looking for a series of peaks that correspond to consecutive members of a fragment series. Each of the mass differences between two neighboring peaks must be equal to one of the 20 amino acid masses. Much of the specificity of a sequence tag in database searches comes from the mass information encoded in the two flanking masses. In this way, even a tag of two or three amino acids is usually unique in the proteome, especially given the very high precursor mass accuracy possible with modern, high-resolution mass spectrometers. A tag sequence that is part of an in silico peptide but with a wrong parent mass points to a novel and potentially interesting ...

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Section: Properties Of Identifiable Tandem Mass Spectrasupporting

confidence: 83%

How much peptide sequence information is contained in ion trap tandem mass spectra?

Cox

Hubner

Mann

2008

J. Am. Soc. Mass Spectrom.

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“…The mass difference (5 Da) in these pairs of ions further confirms their assignment as immonium ions, because they lack the carbonyl carbon atom. Their high intensities are consistent with the presence of a fixed positive charge on a nitrogen atom, 26 and references therein.…”

Section: Identification Of Silac Pairs Corresponding To Biotinylated supporting

confidence: 69%

“…3(B)]. These corresponded to fragment ions of the calculated mass of 310.159 Da for the light biotinylated lysine immonium ion after a loss of ammonia, 26 and an additional species most likely due to oxidation of the biotinylated lysine yielding an ion of calculated mass of 326.154 Da for the light version. Heavy counterparts of these ions are also annotated in Figure 3.…”

Section: Identification Of Silac Pairs Corresponding To Biotinylated mentioning

confidence: 98%

A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization

et al. 2009

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We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N-hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions-at K265 and K481 of PB1, which were about three-and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein-protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs.

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“…An asterisk is used to signify the precursor; triangles represent fragments arising from the AMCA substituent; a superscript o represents the loss of NH 3 or H 2 O; P represents a protonated ASHLGLAR; -AMCA' represents the loss of the AMCA chromophore with the retention of the hydrazine functionality by the peptide (see Figure 1) LQVQLSIR-AMCA, ones that were not observed upon CID due to the low mass cutoff. Immonium ions are frequently detected in hybrid quadrupole-time-of-flight mass spectrometers [47,48], and have proven useful for the determination of amino acid compositional information and the assessment of post-translational modifications [48][49][50].…”

Section: Comparison Of Uvpd and Cid Of Carboxylatederivatized Peptidesmentioning

confidence: 99%

Ultraviolet Photodissociation of Carboxylate-Derivatized Peptides in a Quadrupole Ion Trap

Brodbelt

2011

J. Am. Soc. Mass Spectrom.

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The fragmentation patterns obtained by ultraviolet photodissociation (UVPD) and collisioninduced dissociation (CID) in a quadrupole ion trap mass spectrometer were compared for peptides modified at their C-termini and at acidic amino acids. Attachment of Alexa Fluor 350 or 7-amino-4-methyl-coumarin chromophores at the C-terminal and acidic residues enhances the UV absorptivity of the peptides and all fragment ions that retain the chromophore, such as the y ions that contain the chromophore-modified C-terminus. Whereas CID results in the formation of the typical array of mainly y-type and a/b-type fragment ions, UVPD produces predominantly a/ b-type ions with greatly reduced abundances of y ions. Immonium ions, mostly ones from aromatic or basic amino acids, are also observed in the low m/z range upon UVPD. UVPD of peptides containing two chromophore moieties (with one at the C-terminus and another at an acidic residue) results in even more efficient photodissociation at the expense of the annihilation of almost all diagnostic b and y ions containing the chromophore.

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Collision-induced reporter fragmentations for identification of covalently modified peptides

Cited by 37 publications

References 79 publications

How much peptide sequence information is contained in ion trap tandem mass spectra?

How much peptide sequence information is contained in ion trap tandem mass spectra?

A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization

Ultraviolet Photodissociation of Carboxylate-Derivatized Peptides in a Quadrupole Ion Trap

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