Critical assessment of accelerating trypsination methods.
15 Dec 2011-Journal of Pharmaceutical and Biomedical Analysis (Elsevier)-Vol. 56, Iss: 5, pp 1069-1078
TL;DR: Recommendations regarding optimizing and evaluating the tryptic digestion for both targeted and comprehensive proteomics are given, and a digestion method suitable as the first method for newcomers in Comprehensive proteomics is suggested.
About: This article is published in Journal of Pharmaceutical and Biomedical Analysis.The article was published on 2011-12-15. It has received 59 citations till now. The article focuses on the topics: Protein digestion & Digestion (alchemy).
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24 Feb 2012
TL;DR: The failure in the search for biomarkers as indicators of disease, the difficulties of protein arrays, the uncertainty of quantification in “shotgun proteomics”, database shortcomings, and finally the huge disappointment in the area of drug discovery are addressed.
Abstract: Proteomics is defined as the large-scale study of proteins in particular for their structures and functions (Anderson and Anderson 1998), and investigations of proteins have become very important since they are the main components of the physiological metabolic pathways in eukaryotic cells. Proteomics increasingly plays an important role in areas like protein interaction studies, biomarker discovery, cancer prevention, drug treatment and disease screening medical diagnostics (Capelo et al. 2009). Proteomics can be performed either in a comprehensive or “shotgun” mode, where proteins are identified in complex mixtures, or as “targeted proteomics” where “selective reaction monitoring” (SRM) is used to choose in advance the proteins to observe, and then measuring them accurately, by optimizing the sample preparation as well as the LC-MS method in accordance to the specific proteins (Mitchell 2010). Whether “MS-based shotgun proteomics” has accomplished anything at all regarding clinically useful results was recently addressed by Peter Mitchell in a feature article (Mitchell 2010), and he states that the field needs to make a further step or even change direction. Referring to discussions with among others John Yates and Matthias Mann, Mitchell addresses the failure in the search for biomarkers as indicators of disease, the difficulties of protein arrays, the uncertainty of quantification in “shotgun proteomics” (due to among others the efficiency of ionization in the mass spectrometers), database shortcomings, the problems of detecting post translational modifications (PTMs), and finally the huge disappointment in the area of drug discovery. The field points in the direction of targeted proteomics, but targeted proteomics will not be the solution to all our questions and comprehensive proteomics will still be needed. In order to get as much information, with as high quality as possible, from a biological sample, both the sample preparation and the final LC-MS analyses need to be optimized. The most important step in the sample preparation for proteomics is the conversion of proteins to peptides and in most cases trypsin is used as enzyme. Trypsin is a protease that specifically cleaves the proteins creating peptides both in the preferred mass range for MS sequencing and with a basic residue at the carboxyl terminus of the peptide, producing information-rich, easily interpretable peptide fragmentation mass spectra. Some other proteases can be used as well, such as Lys-C, which is active in more harsh conditions with 8 M urea, and give larger fragments than trypsin. Asp-N and Glu-C are also highly sequence-
91 citations
TL;DR: This chapter contains a nanoscale liquid chromatography-mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG) and describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data.
Abstract: This chapter contains a nanoscale liquid chromatography-mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.After (optional) affinity purification, the pure IgG is cleaved with trypsin. The tryptic glycopeptides are separated almost exclusively on their peptide backbone. This ensures similar response factors for all glycoforms in the MS detection and allows the collection of separate glycoform profiles for different IgG isoforms or allotypes. LaCyTools automatically performs label-free (relative) quantitation of the obtained data after minimal manual input and additionally calculates several quality criteria which can be used for data curation at the level of both individual analytes and entire LC-MS runs.
68 citations
TL;DR: Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry.
Abstract: Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and Ne-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.
67 citations
Cites background from "Critical assessment of accelerating..."
...5 with heating at 50 – 60 °C in some protocols [108]....
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TL;DR: While the performance of individual trypsins across repeat digests was reproducible, significant differences were observed depending on the origin of the trypsin (i.e., bovine vs porcine).
Abstract: Trypsin is an endoprotease commonly used for sample preparation in proteomics experiments. Importantly, protein digestion is dependent on multiple factors, including the trypsin origin and digestion conditions. In-depth characterization of trypsin activity could lead to improved reliability of peptide detection and quantitation in both targeted and discovery proteomics studies. To this end, we assembled a data analysis pipeline and suite of visualization tools for quality control and comprehensive characterization of preanalytical variability in proteomics experiments. Using these tools, we evaluated six available proteomics-grade trypsins and their digestion of a single purified protein, human serum albumin (HSA). HSA was aliquoted and then digested for 2 or 18 h for each trypsin, and the resulting digests were desalted and analyzed in triplicate by reversed-phase liquid chromatography-tandem mass spectrometry. Peptides were identified and quantified using the NIST MSQC pipeline and a comprehensive HSA mass spectral library. We performed a statistical analysis of peptide abundances from different digests and further visualized the data using the principal component analysis and quantitative protein "sequence maps". While the performance of individual trypsins across repeat digests was reproducible, significant differences were observed depending on the origin of the trypsin (i.e., bovine vs porcine). Bovine trypsins produced a higher number of peptides containing missed cleavages, whereas porcine trypsins produced more semitryptic peptides. In addition, many cleavage sites showed variable digestion kinetics patterns, evident from the comparison of peptide abundances in 2 h vs 18 h digests. Overall, this work illustrates effects of an often neglected source of variability in proteomics experiments: the origin of the trypsin.
62 citations
TL;DR: It is shown that even minor differences in the peptide backbone and solvent do significantly influence HILIC glycopeptide enrichment and need to be carefully considered when employed for glycopePTide enrichment.
Abstract: Glycopeptide enrichment is a crucial step in glycoproteomics for which hydrophilic interaction chromatography (HILIC) has extensively been applied due to its low bias towards different glycan types. A systematic evaluation of applicable HILIC mobile phases on glycopeptide enrichment efficiency and selectivity is, to date, however, still lacking. Here, we present a novel, simplified technique for HILIC enrichment termed "Drop-HILIC", which was applied to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type of hydrophilic interaction chromatography) glycopeptide enrichment. The four most commonly used MS compatible organic solvents were investigated: (i) acetonitrile, (ii) methanol, (iii) ethanol and (iv) isopropanol. Glycopeptide enrichment efficiencies were evaluated for each solvent system using samples of increasing complexity ranging from well-defined synthetic glycopeptides spiked into different concentrations of tryptic BSA peptides, followed by standard glycoproteins, and a complex sample derived from human (depleted and non-depleted) serum. ZIC-HILIC glycopeptide efficiency largely relied upon the used solvent. Different organic mobile phases enriched distinct glycopeptide subsets in a peptide backbone hydrophilicity-dependant manner. Acetonitrile provided the best compromise for the retention of both hydrophilic and hydrophobic glycopeptides, whereas methanol was confirmed to be unsuitable for this purpose. The enrichment efficiency of ethanol and isopropanol towards highly hydrophobic glycopeptides was compromised as considerable co-enrichment of unmodified peptides occurred, though for some hydrophobic glycopeptides isopropanol showed the best enrichment properties. This study shows that even minor differences in the peptide backbone and solvent do significantly influence HILIC glycopeptide enrichment and need to be carefully considered when employed for glycopeptide enrichment. Graphical Abstract The organic solvent plays a crucial role in ZIC-HILIC glycopeptide enrichment.
59 citations
Cites background from "Critical assessment of accelerating..."
...This step, however, can also lead to the loss of very hydrophilic glycopeptides [37]....
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References
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TL;DR: A method is described, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics and allows single-run analyses of organelles and an unprecedented depth of proteome coverage.
Abstract: A method, filter-aided sample preparation (FASP) combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, allowing deeper proteomic coverage in a shorter analysis time, using small sample amounts. We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
6,096 citations
TL;DR: Quantitative analysis of protein expression data obtained by high‐throughput methods has led to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo.
Abstract: The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high-throughput methods has led us to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.
1,005 citations
TL;DR: A protocol was developed that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage.
Abstract: Kinetics of in-gel digestion of proteins by modified and native trypsins was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards. The effect of the temperature, enzyme concentration, digestion time, and surface area of gel pieces on the yield of digestion products was characterized. Based on the kinetic data, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage. The accelerated digestion protocol was tested in identification of more than 120 proteins from budding and fission yeasts at the subpicomole level.
373 citations
"Critical assessment of accelerating..." refers background or methods in this paper
...Accelerating methods like for instance heating [8], solvent ffects [9–11], ultrasonic energy [12], infrared (IR) assisted [13–15], icrowave assisted [16–20], and microreactors where the trypsin s immobilized on a solid support [21–24] have been reported....
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...Nevertheless, a modified (like methylated) trypsin which is commercially available, that have an optimum digestion temperature at 60 ◦C, could be more efficient in combination with microwave assisted digestion [8]....
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...The trypsin action can also be evaluated by the mass chromatograms using both the base peak chromatogram (BPC) and total ion chromatogram (TIC), to both check if peptides are generated [8] and the presence of any intact protein [15,20]....
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...[8] J....
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...5 min [8,13,14,17,18]....
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TL;DR: It is found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol, however, SDS can be a serious interference for subsequent mass spectrometry.
Abstract: Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides fro...
312 citations
TL;DR: It is demonstrated that proteins are readily digested by trypsin in the presence of organic solvents such as methanol, acetone, 2-propanol, and acetonitrile to show significantly enhanced digestion for nearly all of the protein components.
Abstract: The rate of protein digestion imposes significant limitations on high-throughput protein identification using mass spectrometry. In this report, we demonstrate that proteins are readily digested by trypsin in the presence of organic solvents such as methanol, acetone, 2-propanol, and acetonitrile. The rates of protein digestion in organic solvents, as indicated by the abundances of digest fragment ions in the mass spectrum, are increased relative to aqueous solution. In addition, amino acid coverage for the analyzed proteins increases in the presence of the organic solvents, and proteins that are resistant to proteolysis are readily digested. For example, a 68% amino acid sequence coverage was attained from a tryptic digest of myoglobin in <5 min from an 80% acetonitrile solution, whereas no digest fragments were detected from a 5 min digestion in an aqueous solution. Moreover, the tryptic digestion of a complex protein mixture in an organic−aqueous solvent system showed significantly enhanced digestion f...
275 citations