protein digestion

Use of ProAlanase Digestion Increases Number of Identified Methylglyoxal (MGO)-Modified Proteins in Whole-Cell Lysates

Posted on by Michele Arduengo
space filling structural model methylglyoxal (MGO)
Methylglyoxal is responsible for post translational protein modifications, that result in advanced glycation endproducts (AGEs), which are associated with aging and disease.

Post-translational modifications (PTM) of proteins are essential for the function of many proteins, but aberrant modification of protein residues also can interfere with protein function. PTMs occur in two ways. Proteins may be modified through the activity of enzymes such as kinases, phosphorylases, glycosylases and others that add or remove specific chemical moieties to amino acid residues. PTMs can also result from non-enzymatic reaction between electrophilic compounds and nucleophilic arginine and lysine residues within a protein. Metabolites and metabolic by products produced during glycolysis, especially glyoxal and methylglyoxal (MGO), are examples of such electrophilic compounds. These compounds can react with arginine and lysine to produce advanced glycation end products (AGEs), which are biomarkers associated with aging and degenerative diseases such as Alzheimer disease, diabetes and others. MGO is also elevated in tumors that have switched from oxidative phosphorylation to glycolysis as their main energy production pathway.

Only limited information is available about site-specific MGO PTMs in mammal cells, and most studies have focused on measuring the amount of MGO modifications in a treatment scenario compared to a control. Donnellan and colleagues recently published work to identify specific MGO protein modifications.  They used a “bottom-up” proteomic analysis of WIL2-NS B lymphoblastoid whole-cell lysates to identify specific MGO-modified proteins. In particular, the group was looking for modifications in proteins that might explain how MGO activity contributes to aneuploidy.

For the study, 100µg of cellular protein extract was reduced with dithiothreitol and then alkylated with chloroacetamide. The sample was diluted to reduce urea concentration. Trypsin Gold was added and samples were digested for 8 hours at 37°C. Digestion was terminated by adding formic acid. For ProAlanase digestion, 20µg of protein was reduced, alkylated and diluted to reduce urea concentration before adding digesting with ProAlanase for 4 hours at 37°C.

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The authors identified 519 MGO-modified proteins.  Most of the modifications were identified in the trypsin digestion reactions; however, ProAlanase digestion increased the number of MGO modifications identified by approximately 25% (with less than 4% of the modification sites being detected in both the ProAlanase and trypsin digestion reactions. The authors suggest that ProAlanase increased sequence coverage to reveal sites not detected in the trypsin digestions. Therefore, they conclude that ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

MGO-modified proteins from the WIL2-NS whole cell lysates included proteins involved in glycolysis, translation initiation, protein folding, mRNA splicing, cell-to-cell adhesion, heat response, nucleosome assembly, protein SUMOylation and the G2/M cell cycle transition. More work to further characterize the sites of these modifications and their potential effects on the function of the modified proteins is ongoing.

Read more about ProAlanase, a new site-specific endoprotease from Promega.

Literature Cited

Donnelian, L et al. (2022) Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress Int. J. Mol. Sci. 23(7), 3689 doi.org/10.3390/ijms23073689

How Can You Improve Protein Digests for Mass Spectrometry Analysis?

Posted on by Gary Kobs
Can predigestion with trypisin (ribbon structure shown) improve protein digests for mass spectrometry analysis?
Can pre-digestion with trypsin improve mass spec analysis?

The trypsin protease cleaves proteins on the carboxyterminus of Arginine (Arg) and Lysine (Lys). This cleavage reaction leaves a positive charge on the C-terminus of the resulting peptide, which enhances mass spectrometry analysis (1,2). Because of this advantage, trypsin has become the most commonly used protease for mass spectrometry analysis. Other proteases which cleave differently from trypsin, yielding complementary data are also used in mass spec analysis: these include Asp-N and Glu-C , which cleave acidic residues, and chymotrypsin which cleaves at aromatic residues. The broad spectrum protease, proteinase K is also used for some proteomic analyses. In a recent study, Dau and colleagues investigated whether sequential digestion with trypsin followed by the complementary proteases could improve protein digests for mass spectrometry analysis.

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Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool

Posted on by Kari Kenefick
The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.
The poster featured in this blog provides background information and data on the development of Rapid Digestion-Trypsin.

Improvements in Protein Bioprocessing

As more and more protein-based therapeutics enter research pipelines, more efficient protocols are needed. In particular, we need better protocols for the characterization of protein structure and function, as well as means of quantitation. One main step in this pipeline, proteolysis of these proteins into peptides, presents a bottleneck and can require optimization of multiple steps including reduction, alkylation and digestion time.

We have developed a new trypsin reagent, Rapid Digestion–Trypsin, that streamlines the protein sample preparation process. This new development significantly reduces the time to achieve proteolysis to about 1 hour, a remarkable improvement over existing overnight sample preparation times.

How Does it Work?

With this new trypsin product, proteolysis is performed at 70°C, incorporating both denaturation and rapid digestion. The protocol can be used with multiple protein types, including pure proteins and complex mixtures, and is compatible with digestion under native, reduced or nonreduced conditions.

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ProteaseMAX: A Surfactant for the Most Complex Mixtures

Posted on by Gary Kobs
Alternate Proteases Cover

Here we provide two examples of “atypical” experiments that take advantage of the properties of the ProteaseMAX™ Surfactant to improve studies involving digestion of complex protein mixtures.

Example 1
Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. A recent publication (1) illustrated a novel strategy using three different approaches  to identify proteins of the exosporium layer of C. difficile spores and complements previous proteomic studies on the entire C. difficile spores.

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Filter-Aided Sample Preparation before Mass Spec Analysis: An Evaluation of FASP and eFASP

Posted on by Gary Kobs

12271ma_800pxFilter-aided sample preparation (FASP) method is used for the on-filter digestion of proteins prior to mass-spectrometry-based analyses (1,2). FASP was designed for the removal of detergents, and chaotropes that were used for sample preparation. In addition, FASP removes components such as salts, nucleic acids and lipids. Akylation of reduced cysteine residues is also carried out on filter, after which protein is proteolyzed by use of trypsin on filter in the optimal buffer of the enzyme. Subsequent elution and desalting of the peptide-rich solution then provides a sample ready for LC–MS/MS analysis.

Erde et al. (3) described an enhanced FASP (eFASP) workflow that included 0.2% DCA in the exchange, alkylation, and digestion buffers,thus enhancing trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. DCA has been reported (4) to improve the efficiency of the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides.

In a recent publication (5) traditional FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). The results indicate that at the protein level, both methods extracted essentially the same number of hydrophobic transmembrane containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes.

The LC–MS/MS results indicate that FASP and eFASP showed no significant differences at the protein level. However, because of the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.

  1. Manza, L. L. et al. (2005) Sample preparation and digestion for proteomic analyses using spin filters Proteomics  5, 1742–74.
  2. Wiśniewski, J. R. et al. (2009) Universal sample preparation method for proteome analysis Nat. Methods 6, 359–62.
  3. Erde, J. et al. (2014) Enhanced FASP (eFASP) to increase proteomic coverage and sample recovery for quantitative proteome experiments. J. Proteome Res. 13, 1885–95.
  4. Lin, Y. et al. (2008) Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins Anal. Biochem.  377, 259–66.
  5. Nel. A. et al. (2015) Comparative Reevaluation of FASP and Enhanced FASP methods by LC-MS/MS/ J Proteome Res. 14, 1637–42.

Asp-N Protease: Applications Update

Posted on by Gary Kobs
TrypsinLysC Page 2

Asp-N, Sequencing Grade, is an endoproteinase that hydrolyzes peptide bonds on the N-terminal side of aspartic and cysteic acid residues: Asp and Cys. Asp-N activity is optimal in the pH range of 4.0–9.0. This sequencing grade enzyme can be used alone or in combination with trypsin or other proteases to produce protein digests for peptide mapping applications or protein identification by peptide mass fingerprinting or MS/MS spectral matching. It is suitable for  in-solution or in-gel digestion reactions.

The following references illustrate the use of Asp-N in recent publications:

Protein sequence coverage

  1. Jakobsson, M et al. (2013)  Identification and characterization of a novel Human Methyltransferase modulating Hsp70 protein function through lysine methylation. J. Biol. Chem. 288, 27752–63.
  2. Carroll, J. et. al. (2013) Post-translational modifications near the quinone binding site of mammalian complex I.  J. Biol. Chem. 288, 24799–08.

Glycoprotein analysis

  1. Siguier, B. et al. (2014) First structural insights into α-L-Arabinofuranosidases from the two GH62 Glycoside hydrolase subfamilies. J. Biol. Chem. 289, 5261–73.
  2. Vakhrushev, S. et al. (2013) Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells. Mol. Cell. Prot. 12, 932–44.
  3. Berk, J. et al. (2013) . O-Linked β-N- Acetylglucosamine (O-GlcNAc) Regulates emerin binding to autointegration Factor (BAF) in a chromatin and Lamin B-enriched “Niche”.  J. Biol. Chem. 288, 30192–09.

Phosphoprotein analysis

  1. Roux, P. and Thibault, P. (2013) The Coming of Age of phosphoproteomics –from Large Data sets to Inference of protein Functions. Mol. Cell. Prot. 12, 3453–64.

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Trypsin/Lys-C Mix: Alternative for standard trypsin protein digestions

Posted on by Gary Kobs

Trypsin/Lys-C Mix, Mass Spec Grade, is a mixture of Trypsin Gold, Mass Spectrometry Grade, and rLys-C, Mass Spec Grade. The Trypsin/Lys-C Mix is designed to improve digestion of proteins or protein mixtures in solution.It is a little known fact that trypsin cleaves at lysine residues with lesser efficiency than at arginine residues. Inefficient proteolysis at lysine residues is the major cause of missed (undigested) cleavages in trypsin digests.

11788MA


Supplementing trypsin with Lys-C enables cleavage at lysines with excepetional efficiency and specificity. Following the conventional trypsin digestion protocol (i.e., overnight incubation at nondenaturing conditions, reduction,alkylation, 25:1 protein:protease ratio [w/w], mix and incubate overnight at 37°C.) Replacing trypsin with Trypsin/Lys-C Mix in this conventional protocol leads to multiple benefits for protein analysis including more accurate mass spectrometry-based protein quantitation and improved protein mass spectrometry analytical reproducibility.

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ProteaseMAX Surfactant: Enhanced In-solution Digestion Applications

Posted on by Gary Kobs
ProteaseMax 11228MA

The primary advantage of ProteaseMAX™ Surfactant is that it improves identification of proteins in gel by enhanced protein digestion, increased peptide extraction, and minimized post digestion peptide loss. However, ProteaseMAX™ Surfactant can also facilitate in-solution digestion protocols.

ProteaseMAX™ Surfactant offers two major benefits for digesting proteins in solution.

Continue reading “ProteaseMAX Surfactant: Enhanced In-solution Digestion Applications”

Proteinase K: An Enzyme for Everyone

Posted on by Gary Kobs
protein expression purification and analysis

We recently posted a blog about Proteinase K, a serine protease that exhibits broad cleavage activity produced by the fungus Tritirachium album Limber. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples. In that previous blog we focused on its use to remove RNase and DNase activities. However, the stability of Proteinase K in urea and SDS and its ability to digest native proteins make it useful for a variety of applications. Here we provide a brief list of peer-reviewed citations that demonstrate the use of proteinase K in DNA and RNA purification, protein digestion in FFPE tissue samples, chromatin precipitation assays, and proteinase K protection assays:

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Use of Multiple Proteases for Improved Protein Digestion

Posted on by Gary Kobs

One of the approaches to identify proteins by mass spectrometry includes the separation of proteins by gel electrophoresis or liquid chromatography. Subsequently the proteins are cleaved with sequence-specific endoproteases. Following digestion the generated peptides are investigated by determination of molecular masses or specific sequence. For protein identification the experimentally obtained masses/sequences are compared with theoretical masses/sequences compiled in various databases.

Trypsin is the favored enzyme for this application, for the following reasons: A) the peptides contain a basic residue (Arg or Lys) on the C terminus and thus are good candidates for collision induced activation (CAD) in tandem experiments (low charge states and high mass-to-charge ratios); B) it is relatively Inexpensive; and C) optimal digestion conditions have been well characterized.

An inherent limitation of trypsin is the size of the peptides that it generates. For most organisms > 50% of tryptic peptides are less than 6 amino acids, too small for mass spectrometry based sequencing.

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One recent publication examined the use of multiple proteases (trypsin, LysC, ArgC , AspN and GluC) in combination with either CAD or electron-based fragmentation (ETD) to improve protein identification (1). Their results indicated a significant improvement from a single protease digestion (trypsin), which yielded 27,822 unique peptides corresponding to 3313 proteins. In contrast using a combination of proteases with either CAD or ETD fragmentation methods yielded 92,095 unique peptides mapping to 3908 proteins.

Swaney DL, Wenger CD, & Coon JJ (2010). Value of using multiple proteases for large-scale mass spectrometry-based proteomics. Journal of proteome research, 9 (3), 1323-9 PMID: 20113005