Development of Improved High-Performance Liquid Chromatography Conditions for Nonisotopic Detection of Isoaspartic Acid to Determine the Extent of Protein Deamidation

Carlson, A.D.; Riggin, R.M.

doi:10.1006/abio.1999.4421

Cited by 30 publications

(13 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…First, the low-level amount of proteins typically obtained from biological samples is a practical limit to this and most°quantitation°methods°and°may°prohibit°the°use°of the method entirely or require extensive chromatographic cleanup procedures. Other methods may be necessary,°such°as°immunological° [48]°or°radioactive [25]°methods,°but°these°methods°can°only°detect°isoas-partyl residues, and cannot localize or quantitate the relative abundance of deamidation products. Second, as proteins become larger, their digestion mixtures become more complex.…”

Section: Resultsmentioning

confidence: 99%

“…Radioactive methods utilize the enzyme protein isoaspartyl methyl transferase (PIMT) to selectively methylate isoAsp residues in peptides and proteins with isotopically labeled methyl groups, thus detecting deamidation, but not localizing the site or determining the relative abundance of the products [24,25]. Separation of proteins from their deamidated counterparts can be done by ion-exchange HPLC [23,26,27] or IEF gel electrophoresis [14,28] because there is a net change in the isoelectric point with deamidation.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Cournoyer

Lin

Bowman

et al. 2007

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Relative quantitation of aspartyl and isoaspartyl residue mixtures from asparagine deamidation is demonstrated using electron capture dissociation without prior HPLC separation. The method utilizes the linear relationship found between the relative abundance of the isoaspartyl diagnostic ion, z n -57, and % isoaspartyl content based on the ECD spectra of known isoaspartyl/aspartyl mixtures of synthetic peptides. The observed linearity appears to be sequence independent because the relationship exists despite sequence variations and changes in backbone fragment abundances when isoaspartyl and aspartyl residues are interchanged. Furthermore, a new method to calculate the relative abundances of isomer from protein deamidation without synthetic peptides is proposed and tested using a linear peptide released by protein digestion that contains the deamidation site. The proteolytic peptide can be rapidly aged to the expected 3:1 (isoaspartyl:aspartyl) mixture to generate a two-point calibration standard for ECD analysis. The procedure can then be used to determine the relative abundance of deamidation products from in vivo or in vitro protein aging experiments. (J Am Soc Mass Spectrom 2007, 18, 48 -56)

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Cournoyer

Lin

Bowman

et al. 2007

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…Other assays have been developed for the detection of AdoHcy, but not for the expressed purpose of measuring in vitro PRMT activity [45,46]. Adaptation of these assays to the study of PRMT activities should be relatively straightforward.…”

Section: Methods Of Measuring the Activities Of Prmtsmentioning

confidence: 99%

“…Adaptation of these assays to the study of PRMT activities should be relatively straightforward. For example, the activity of protein isoaspartyl methyltransferase has been measured by quantification of AdoHcy using HPLC [45,46]. As with all assays used for AdoHcy detection that we have discussed, persistent background signals can be problematic and must be addressed accordingly [46].…”

Section: Methods Of Measuring the Activities Of Prmtsmentioning

confidence: 99%

Approaches to measuring the activities of protein arginine N-methyltransferases

Lakowski

Zurita-Lopez²,

Clarke³

et al. 2010

Analytical Biochemistry

View full text Add to dashboard Cite

Despite the emerging importance of protein arginine N-methyltransferase (PRMT) activity in regulating cellular processes, only a limited number of PRMT assays have been developed. Here, we compare several qualitative and quantitative methods that we use for measuring PRMT activity. Gel-based methods allow for the simultaneous detection of methyl transfer activity on multiple substrates, but require signals well above background in order to generate reliable data for quantitation, which can be challenging with low activity PRMTs or substrates that are poor methyl-acceptors. Techniques that measure S-adenosyl-L-homocysteine (AdoHcy) product formation suffer from a background caused by PRMT automethylation and the spontaneous formation of AdoHcy from S-adenosyl-L-methionine (AdoMet). However, when this background is controlled, this approach is useful for product inhibition studies. Methods that detect methylated arginines derived from acid hydrolysis of PRMT reaction samples can determine the absolute amounts of ω-NG-monomethylarginine (MMA), asymmetric ω-NG,NG-dimethylarginine (aDMA) or symmetric ω-NG,N′G-dimethylarginine (sDMA) to quantify PRMT activity. We describe separation methods of these methylated arginine derivatives by thin layer, reverse phase, or cation exchange chromatography, and quantification by radioactivity or mass spectrometry. The latter approach is advantageous because it does not require radiolabelled samples for detection, and activity is readily quantified with commercially available standards.

show abstract

“…Although determining the conversion of Asn to Asp/isoAsp is relatively straightforward, distinguishing the products Asp and isoAsp is more challenging. Several methods for the Asp/isoAsp differentiation exist, including NMR [20], HPLC [21], Edman-based sequencing [22], and antibody detection [23], although they all have certain limitations: the former three typically require relatively large quantity of proteins and the latter requires highly specific antibodies.…”

mentioning

confidence: 99%

Use of ¹⁸O labels to monitor deamidation during protein and peptide sample processing

Cournoyer

Lin

et al. 2008

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c · ϩ 58 and z Ϫ 57 fragment ions for the isoAsp residue and the fragment ion ((M ϩ nH) (nϪ1)ϩ· Ϫ 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18 O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a ϩ3 Da (18 O 1 ϩ 1D) mass shift from the pre-existing deamidation, which leads to a ϩ1-Da mass shift. This paper demonstrates the use of 18 O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (˜4 h) trypsin digestion process generates no additional detectable peptide deamidations. (J Am Soc Mass Spectrom 2008, 19, 855-864)

show abstract

Development of Improved High-Performance Liquid Chromatography Conditions for Nonisotopic Detection of Isoaspartic Acid to Determine the Extent of Protein Deamidation

Cited by 30 publications

References 14 publications

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Approaches to measuring the activities of protein arginine N-methyltransferases

Use of ¹⁸O labels to monitor deamidation during protein and peptide sample processing

Contact Info

Product

Resources

About

Development of Improved High-Performance Liquid Chromatography Conditions for Nonisotopic Detection of Isoaspartic Acid to Determine the Extent of Protein Deamidation

Cited by 30 publications

References 14 publications

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Approaches to measuring the activities of protein arginine N-methyltransferases

Use of 18O labels to monitor deamidation during protein and peptide sample processing

Contact Info

Product

Resources

About

Use of ¹⁸O labels to monitor deamidation during protein and peptide sample processing