Reliable Detection of Deamidated Peptides from Lens Crystallin Proteins Using Changes in Reversed-Phase Elution Times and Parent Ion Masses

Dasari, Surendra; Wilmarth, Phillip A.; Rustvold, D. Leif; Riviere, Michael A.; Nagalla, Srinivasa R.; David, Larry L.

doi:10.1021/pr070182x

Cited by 33 publications

(41 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fig. 2 shows that the deamidated peptide DGEA-GAQ*GPPGPAGPAGER (COL1A1-0441-0 Van Doorn et al nomenclature (58)) can be distinguished by the MS ion envelope and elutes after the non-deamidated form (59). Interestingly, peptides derived from noncollagen proteins showed a very high to complete level of deamidation.…”

Section: Resultsmentioning

confidence: 99%

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Hill

Wither

Nemkov

et al. 2015

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ϳ120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in wellpreserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. During the last decade, paleontology and taphonomy (the study of decaying organisms over time and the fossilization processes) have begun to overlap with the field of proteomics to shed new light on preserved organic matter in fossilized bones (1-4). These bones represent a time capsule of ancient biomolecules, owing to their natural resistance to post mortem decay arising from a unique combination of mechanical, structural, and chemical properties (4 -7).Although bones can be cursorily described as a composite of collagen (protein) and hydroxyapatite (mineral), fossilized bones undergo three distinct diagenesis pathways: (i) chemical deterioration of the organic phase; (ii) chemical deterioration of the mineral phase; and (iii) (micro)biological attack of the composite (6). In addition, the rate of these degradation pathways are affected by temperature, as higher burial temperatures have been shown to accelerate these processes (6,8). Though relatively unusual, the first of these three pathways results in a slower deterioration process, which is more generally mitigated under (6) specific environmental constraints, such as geochemical stability (stable temperature and acidity) that promote bone mineral preservation. Importantly, slower deterioration results in more pre...

show abstract

Section: Resultsmentioning

confidence: 99%

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Hill

Wither

Nemkov

et al. 2015

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…1D, an amidated peptide and its 13 C peak have the same retention time in RP chromatography, but a deamidated peptide has a different retention time than that of the corresponding amidated peptide. This has been used in the reliable identification of deamidated peptides (19,29). However, the method that we propose here is decidedly more convenient because it can be achieved easily by merely changing the parameters in database searches.…”

Section: Resultsmentioning

confidence: 99%

Detection, Evaluation and Minimization of Nonenzymatic Deamidation in Proteomic Sample Preparation

Hao

Ren

Alpert³

et al. 2011

Molecular & Cellular Proteomics

148

188

View full text Add to dashboard Cite

Identification of deamidated sites in proteins is commonly used for assignment of N-glycosylation sites. It is also important for assessing the role of deamidation in vivo. However, nonenzymatic deamidation occurs easily in peptides under conditions commonly used in treatment with trypsin and PNGase F. The impact on proteomic sample preparation has not yet been evaluated systematically. In addition, the 13 C peaks of amidated peptides can be misassigned as monoisotopic peaks of the corresponding deamidated ones in database searches. The 19.34 mDa mass difference between them is proposed as a means for eliminating the resulting false positive identifications in large-scale proteomic analysis. We evaluated five groups of proteomic data, obtained mainly through an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)-reverse phase (RP) chromatography sequence, and ascertained that nonenzymatic asparagine deamidation occurred to some extent on 4 -9% of the peptides, resulting in the false positive identification of many N-glycosylation sites. A comprehensive investigation indicated that the chief causative factors were the mildly alkaline pH and prolonged incubations at 37°C during proteomic sample preparation. An improved protocol is proposed featuring tryptic digestion at pH 6 and deglycosylation at pH 5, resulting in a significant decrease in nonenzymatic deamidation while conserving adequate digestion efficiency. The number of identified deamidation sites was improved significantly by increasing the sample loading amount in liquid chromatography-tandem MS. This permitted the identification of a significant number of glutamine deamidation sites, which featured sequence motifs largely different from those for asparagine deami- Accurate identification of protein deamidation sites is important to understanding the role of deamidation in human diseases and aging and in the correct location of N-glycosylation using the PNGase F deglycosylation method. However, determination of protein deamidation with liquid chromatography-tandem MS (LC-MS/MS) 1 is compromised by current methods of proteomics sample preparation and database searching. Nonenzymatic deamidation of Asn and Gln occurs spontaneously on proteins and peptides both in vivo and in vitro. In vivo, deamidation is reported to be related to Alzheimer's disease and cataracts (1-3). It has also been proposed as a molecular clock in studies of aging (4, 5). In vitro, deamidation leads to the degradation of therapeutic proteins during production and storage (6, 7). It takes place relatively slowly in intact proteins with a half-life of 1-500 days for Asn and 100 -500 days for Gln (8). However, the deamidation rate of peptides increases significantly under conditions typical of tryptic digestion, i.e. incubation in digestion buffers at pH 8 for 8 -16 h at 37°C (9). Under mildly alkaline conditions, Asn deamidation happens mainly through the formation of a succinimide ring intermediate that is quickly hydrolyzed to D,LAsp and D,L-isoAsp with isoAsp pred...

show abstract

“…Conversion of an amide to a carboxylic acid causes a 0.984 Da mass increase, which is almost identical to the mass of the non-deamidated peptide having an extra neutron from naturally occurring heavy isotopes of the constitute elements. Since mass spectrometers can accurately measure the monoistopic masses of peptides, it is easy to distinguish between non-deamidated and deamidated forms if the resulting chromatographic peaks of the two peptides forms are chromatographically resolved from one another (Dasari et al, 2007). Unfortunately, this is not always possible.…”

Section: Challenges Of Detecting and Quantifying Deamidationmentioning

confidence: 99%

Lens β-crystallins: The role of deamidation and related modifications in aging and cataract

Lampi¹,

Wilmarth²,

Murray³

et al. 2014

Progress in Biophysics and Molecular Biology

Self Cite

126

117

View full text Add to dashboard Cite

Crystallins are the major proteins in the lens of the eye and function to maintain transparency of the lens. Of the human crystallins, α, β, and γ, the β-crystallins remain the most elusive in their structural significance due to their greater number of subunits and possible oligomer formations. The β-crystallins are also heavily modified during aging. This review focuses on the functional significance of deamidation and the related modifications of racemization and isomerization, the major modifications in β-crystallins of the aged human lens. Elucidating the role of these modifications in cataract formation has been slow, because they are analytically among the most difficult post-translational modifications to study. Recent results suggest that many amides deamidate to similar extent in normal aged and cataractous lenses, while others may undergo greater deamidation in cataract. Mimicking deamidation at critical structural regions induces structural changes that disrupt the stability of the β-crystallins and lead to their aggregation in vitro. Deamidations at the surface disrupt interactions with other crystallins. Additionally, the α-crystallin chaperone is unable to completely prevent deamidated β-crystallins from insolubilization. Therefore, deamidation of β-crystallins may enhance their precipitation and light scattering in vivo contributing to cataract formation. Future experiments are needed to quantify differences in deamidation rates at all Asn and Gln residues within crystallins from aged and cataractous lenses, as well as racemization and isomerization which potentially perturb protein structure greater than deamidation alone. Quantitative data is greatly needed to investigate the importance of these major age-related modifications in cataract formation.

show abstract

Reliable Detection of Deamidated Peptides from Lens Crystallin Proteins Using Changes in Reversed-Phase Elution Times and Parent Ion Masses

Cited by 33 publications

References 28 publications

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Detection, Evaluation and Minimization of Nonenzymatic Deamidation in Proteomic Sample Preparation

Lens β-crystallins: The role of deamidation and related modifications in aging and cataract

Contact Info

Product

Resources

About