An Improved Protocol for N-Glycosylation Analysis of Gel-Separated Sialylated Glycoproteins by MALDI-TOF/TOF

Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots

Simultaneous Analysis of Proteome, Phospho- and Glycoproteome of Rat Kidney Tissue with Electrostatic Repulsion Hydrophilic Interaction Chromatography

Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation

Development and application of novel proteomics methods in proteomics research

The sensitivity, throughput and accuracy of proteomics have improved considerably in the past years due to the development of novel technologies. However, characterization of protein post-translational modifications remains challenging, especially those less studied ones. Protein deamidation is reported to be involved in aging and many diseases, but so far is less studied due to technical challenges. The existing challenges for accurately identifying and quantifying protein deamidation sites in proteomics include tremendous false positive identifications of deamidated peptides from database searches, substantial interference of artificial deamidation from sample preparation, difficulties in differentiating and quantifying the isomeric n-Asp and isoAsp deamidation products in proteome scale, and inability of validating quantification of deamidated peptides by either ELISA or multiple reaction monitoring. In this thesis, novel proteomic methods were developed to overcome these challenges and facilitate the large-scale characterization of protein deamidation. The accurate and reliable characterization of endogenous protein deamidation becomes feasible with both false positive identifications and artificial deamidation minimized while using our new data analysis strategy and sample preparation protocols. High-resolution parallel reaction monitoring enables the accurate validation of deamidated peptides in unfractionated individual samples. In addition to the validation of these methods using both model proteins and complex tissue samples, we also applied them to the study of protein deamidation in human carotid atherosclerotic plaques from aged patients for predicting secondary cerebrovascular events. The biomarker candidates of deamidated peptides and proteins obtained from label-free quantification on pool samples were validated in 38 individual plaques using parallel reaction monitoring for their applicability as biomarkers. None of them reached the criterion as a clinically usable biomarker possibly due to the small sample size and high individual variation. However, these newly developed technologies open a door for scientists who are interested in understanding the role of protein deamidation in aging and many diseases.DOCTOR OF PHILOSOPHY (SBS

The Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) for Proteomics Research

Characterization and studies of proteome are challenging because biological samples are complex, with a wide dynamic range of abundance. At present the proteins are identified by digestion into peptides, with subsequent identification of the peptides by mass spectrometry (MS). MS is a powerful technique for the purpose, but it cannot identify every peptide in such complex mixtures simultaneously. For accurate analysis and quantification it is important to separate the peptides first by chromatography into fractions of a size that MS can handle. With these less complex fractions, the probability is increased of identifying peptides of low abundance that would otherwise experience ion suppression effects due to the presence of peptides of high abundance. Enrichment for peptides with certain post-translational modifications helps to increase their detection rates as well. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is a mixed-mode chromatographic technique which combines the use of electrostatic repulsion and hydrophilic interaction. This review provides an overview of ERLIC and its various proteomics applications. ERLIC has been demonstrated to have good orthogonality to reverse phase liquid chromatography (RPLC), making it useful as a first dimension in multidimensional liquid chromatography (MDLC) and fractionation of digests in general. Peptides elute in order of their isoelectric points and polarity. ERLIC has also been successfully utilized for the enrichment for phosphopeptides and glycopeptides, facilitating their identification. In addition, it is promising for the study of peptide deamidation. ERLIC performs comparably well or better than established methods for these various applications, and serves as a viable and efficient workflow alternative.NMRC (Natl Medical Research Council, S’pore)Published versio

Correction of errors in tandem mass spectrum extraction enhances phosphopeptide identification

The tandem mass spectrum extraction of phosphopeptides is more difficult and error-prone than that of unmodified peptides due to their lower abundance, lower ionization efficiency, the cofragmentation with other high-abundance peptides, and the use of MS3 on MS2 fragments with neutral losses. However, there are still no established methods to evaluate its correctness. Here we propose to identify and correct these errors via the combinatorial use of multiple spectrum extraction tools. We evaluated five free and two commercial extraction tools using Mascot and phosphoproteomics raw data from LTQ FT Ultra, in which RawXtract 1.9.9.2 identified the highest number of unique phosphopeptides (peptide expectation value <0.05). Surprisingly, ProteoWizzard (v. 3.0.3476) extracted wrong precursor mass for most MS3 spectra. Comparison of the top three free extraction tools showed that only 54% of the identified spectra were identified consistently from all three tools, indicating that some errors might happen during spectrum extraction. Manual check of 258 spectra not identified from all three tools revealed 405 errors of spectrum extraction with 7.4% in selecting wrong precursor charge, 50.6% in selecting wrong precursor mass, and 42.1% in exporting MS/MS fragments. We then corrected the errors by selecting the best extracted MGF file for each spectrum among the three tools for another database search. With the errors corrected, it results in the 22.4 and 12.2% increase in spectrum matches and unique peptide identification, respectively, compared with the best single method. Correction of errors in spectrum extraction improves both the sensitivity and confidence of phosphopeptide identification. Data analysis on nonphosphopeptide spectra indicates that this strategy applies to unmodified peptides as well. The identification of errors in spectrum extraction will promote the improvement of spectrum extraction tools in future.NMRC (Natl Medical Research Council, S’pore)Accepted versio

Relative abundance (%) of the glycans identified at Asn107 of human α1-antitrypsin ^a.

a<p>Approximate abundance was estimated using MS signal intensities from a single analysis.</p

MS and MS/MS spectra of glycopeptides from 10 pmol gel-separated human transferrin.

<p>(A) Mass spectrum of the glycopeptides of human transferrin. (B) The enlarged view of Figure 2A, which shows low-abundance glycopeptides more clearly. (C) Mass spectrum of the parent ion of m/z 3684.6 obtained in TOF/TOF mode. The difference between 3682.3, 3391.6 and 3100.5 is about 291. (D) MS/MS spectrum from the precursor indicated by arrow in Figure 2C. The mass of [M_pep+H]⁺ was 1476.7. Glycan structures were deduced based on the difference between adjacent signals and its biosynthesis process.</p

MS and MS/MS spectra of deglycosylated peptides and desialylated glycans of human transferrin.

<p>(A) Mass spectrum of deglycosylated peptides of human transferrin, m/z 1477.681, 2516.072 and 3530.644 were identified as deglycosylated peptides. (B) MS/MS spectrum of m/z 2516 indicated by arrow in Figure 3A. (C) Mass spectrum of desialylated glycans of human transferrin. The MS signals were [M+Na]⁺ ions in average values, and corresponding structures were also shown.</p