1. Overview of Post-Translational Modifications (PTMs) Analysis:
PTMs(hereafter): Phosphorylation (pS/T, pY), Methylation, Deamidation, Oxidation, Nitration, N-glycosylation, Amino acid mutation, Unnatural amino acid, Chemical modifications, Palmitoylation, Glycosylation, Ubiquitination, SUMOylation, Dimethylation, Acetylation, Decarboxylation, etc..
1) Significance:
Protein post-translational modifications play a key role in many cellular processes such as cellular differentiation (Grotenbreg and Ploegh, 2007), protein degradation (Geiss-Friedlander and Melchior, 2007), signaling and regulatory processes (Morrison, et al 2002), regulation of gene expression, and protein-protein interactions. These modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis and influence almost all aspects of normal cell biology and pathogenesis. Therefore, identifying and understanding PTMs is critical in the study of cell biology and disease treatment and prevention.
Post-translational modifications are key mechanisms to increase proteomic diversity.
While the human genome comprises 20-25,000 genes, the proteome is estimated to encompass over 1 million proteins. Changes at the transcriptional and mRNA levels increase the size of the transcriptome relative to the genome, and the myriad of different post-translational modifications exponentially increases the complexity of the proteome relative to both the transcriptome and genome.
2) Procedures:
a) Digesting the proteins into peptides
b) Analyzing the peptides using tandem mass spectrometry (LC-MS/MS)
c) Searching for modifications using SEQUEST, or specialized PTM software
d) Verifying the modification by hand
e) Our opinion of their likelihood of being correct along with the scores from the specific programs we use.
3) Matters need attention:
a) Some Modifications (Phosphorylations, etc.) are easier to find than others. We can look for specific modifications or unknown modifications.
b) As a general rule, any post-translational modification (PTM) could be searched for in your protein as long as we know the mass added by the modification and the potentially modified amino acid (e.g. in the case of phosphorylation: +80 Da on a Serine, Threonine or Tyrosine).
c) We cannot distinguish two modifications of the same nominal mass (e.g. trimethylation [+42 Da] vs. acetylation [+42 Da]).
d) If you can send us your protein sequence and the location of the site of interest, it will be helpful for us to confirm a known modification site or to verify a potential modification site. Since a modified peptide is much less abundant than its non-modified counterparts, we need to preferentially target the modified peptides’ masses for fragmentation according to your information, and increase the probability of acquiring an MS/MS spectrum for these peptides.
4) Sample amount:
As modification stoichiometry is often low, a larger quantity of protein to be analyzed would be desirable for post-translational modifications analysis.
5) Enrichment strategies:
Fixed mass modifications, stable in proteins, include protein acetylation, ubiquitination, methylation and phosphorylation.
The most important consideration in characterizing PTMs is their frequency of occurrence. Since most PTMs are rare events, specific biochemical enrichment strategies need to be used to facilitate their identification and characterization.
a) For phospho peptides: Metal based resins (IMAC) and TiO2 column under acidic conditions are commonly used for the enrichment of phospho peptides before mass spectrometry analysis.
In the approach of using TiO2 column, phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO2 column (GL Sciences, Tokyo, Japan) while nonphosphorylated peptides are washed out. Subsequently, phosphopeptides are desorbed from the TiO2 column under alkaline conditions, and analyzed by nanoflow LC- MS/MS.
In the case of complex biological samples, we utilize multi-dimensional fractionation techniques to reduce the complexity of peptide mixtures prior subjecting samples to mass spectrometric analysis. In the first dimension, samples are fractionated by off line strong cation exchange chromatography (SCX) into 8-10 fractions. Each fraction enriched by TiO2 column and subjected to a further separation using reversed phase chromatography in the second dimension following by mass spectrometry.
b) For tyrosine phosphorylated proteins: Immunoprecipitate with anti-phosphotyrosine antibodies can enrich low-abundant tyrosine phosphorylated proteins from the complex mixtures of proteins such as cell lysates. However, these antibodies are not very good at enriching for phosphopeptides. Also, there are no antibodies for enriching proteins that are phosphorylated on serine or threonine residues.
c) For other modifications: Using specific antibody.
2. PTM Profiling
This service uses multiple enzymes to maximize the sequence coverage obtained for your protein of interest. This approach increases the likelihood of detecting PTMs on your protein including those on none tryptic peptides. The range of modifications is limited to those readily identifiable in a database search, such as methylation of lysine or arginine, ubiquitination of lysine, deamidation of glutamine or asparagine, glycation of lysine, phosphorylation of serine, threonine or tyrosine (without enrichment), or acetylation of lysine.
1) Phosphoproteomics
Protein phosphorylation is a ubiquitous and very important post-translational modification (PTM), which affects an estimated one third of all proteins. Many critical events involved in cellular response are mediated by phosphorylation and dephosphorylation, such as regulation of enzymatic activity, protein conformational change, protein-protein interaction, and cellular localization. Abnormal phosphorylation often leads to severe diseases.
Phosphoproteomics refers to the global analysis of the phosphate modified proteome. Typically phosphorylated amino acids include serine, threonine, and tyrosine and we have established methods to enrich and analyze peptides containing these modified residues. For phospho-serine/threonine peptides we use a combination of ion exchange chromatography and titanium dioxide enrichment prior to LC/MS. For phosphotyrosine containing peptides immunoaffinity capture is used.
a) Guidelines for Identification of Phosphate Modifications
i. Proteins being investigated for phosphorylations should be analyzed as soon as possible after isolation and purification, to minimize possible loss of labile phosphate modifications. If the protein must be stored, -80oC is recommended.
ii. Selective purification of phosphopeptides can be performed at Creative Proteomics using IMAC (immobilized metal affinity chromatography) or TiO2 columns. Roughly 5 or more pmoles of protein is required for IMAC purification.
iii. TiO2 is a new technique that is perhaps 10 to 100 times more sensitive for selective purification of phosphopeptides, compared to IMAC. (The TiO2 purification technique will be available soon).
iv. Phosphopeptides and the exact site of modification can be identified from protein digests (i.e. in-gel or solution), nanoLC/MS/MS, and database searches (specifically for phosphate modification)
v. Phosphorylations can be identified from analysis of intact proteins. Although the site of modification will not characterized from an intact protein analysis, the relative extent and number of phosphorylations can be determined.
vi. Phosphate buffers cannot be used in protein solutions where identification of phosphorylated peptides will be performed using IMAC purification.
vii. EDTA, flag-tag peptide, and other acidic compounds, should not be used in protein solutions where identification of phosphorylated peptides
will be performed using IMAC purification. Unbound flag tag peptide in solution must be removed before IMAC purification.
viii. For identification of phosphorylations and other post-translational modifications, it is recommended that the protein be analyzed from solution (if the solution is suitable), since in principle, all peptides products from digestion of the protein will be injected during nanoLC/MS/MS analysis.
ix. Protein digests with chymotrypsin may result in loss of phosphorylation (there may be evidence of some phosphatase activity in chymotrypsin).
x. The analysis of phosphoproteins is still one of the most challenging tasks in proteome research for a few reasons. Specifically, only a small fraction of the available intracellular pool of a protein is phosphorylated at any given time. The phosphorylated sites on proteins might vary due to the fact that many phosphoproteins exist in several different phosphorylated forms. Finally, most analytical techniques used for studying protein phosphorylation have a limited dynamic range, and in the complex biological mixtures minor phosphorylated sites might be difficult to identify.
2) Glycosylation
Protein glycosylation is acknowledged as one of the major post-translational modifications, with significant effects on protein folding, conformation, distribution, stability and activity. More than half of all mammalian proteins are known to be glycosylated.
Glycosylation encompasses a diverse selection of sugar-moiety additions to proteins that ranges from simple monosaccharide modifications of nuclear transcription factors to highly complex branched polysaccharide changes of cell surface receptors. Carbohydrates in the form of aspargine-linked (N-linked) or serine/threonine-linked (O-linked) oligosaccharides are major structural components of many cell surface and secreted proteins.
3) Ubiquitination
Ubiquitin is an about 8.5 kDa polypeptide consisting of 76 amino acids that is appended to the ε-NH2 of lysine in target proteins via the C-terminal glycine of ubiquitin. Following an initial monoubiquitination event, the formation of a ubiquitin polymer may occur, and polyubiquitinated proteins are then recognized by the 26S proteasome that catalyzes the degradation of the ubiquitinated protein and the recycling of ubiquitin.
4) S-Nitrosylation
Nitric oxide (NO) is produced by three isoforms of nitric oxide synthase (NOS) and is a chemical messenger that reacts with free cysteine residues to form S-nitrothiols (SNOs). S-nitrosylation is a critical PTM used by cells to stabilize proteins, regulate gene expression and provide NO donors, and the generation, localization, activation and catabolism of SNOs are tightly regulated.
S-nitrosylation is a reversible reaction, and SNOs have a short half life in the cytoplasm because of the host of reducing enzymes, including glutathione (GSH) and thioredoxin, that denitrosylate proteins. Therefore, SNOs are often stored in membranes, vesicles, the interstitial space and lipophilic protein folds to protect them from denitrosylation. For example, caspases, which mediate apoptosis, are stored in the mitochondrial intermembrane space as SNOs. In response to extra- or intracellular cues, the caspases are released into the cytoplasm, and the highly reducing environment rapidly denitrosylates the proteins, resulting in caspase activation and the induction of apoptosis.
S-nitrosylation is not a random event, and only specific cysteine residues are S-nitrosylated. Because proteins may contain multiple cysteines and due to the labile nature of SNOs, S-nitrosylated cysteines can be difficult to detect and distinguish from non-S-nitrosylated amino acids. The biotin switch assay, developed by Jaffrey et al., is a common method of detecting SNOs, and the steps of the assay are listed below:
a) All free cysteines are blocked.
b) All remaining cysteines (presumably only those that are denitrosylated) are denitrosylated.
c) The now-free thiol groups are then biotinylated.
d) Biotinylated proteins are detected by SDS-PAGE and Western blot analysis or mass spectrometry.
5) Methylation
The transfer of one-carbon methyl groups to nitrogen or oxygen (N- and O-methylation, respectively) to amino acid side chains increases the hydrophobicity of the protein and can neutralize a negative amino acid charge when bound to carboxylic acids. Methylation is mediated by methyltransferases, and S-adenosyl methionine (SAM) is the primary methyl group donor.
Methylation occurs so often that SAM has been suggested to be the most-used substrate in enzymatic reactions after ATP. Additionally, while N-methylation is irreversible, O-methylation is potentially reversible.
Methylation is a well-known mechanism of epigenetic regulation, as histone methylation and demethylation influences the availability of DNA for transcription. Amino acid residues can be conjugated to a single methyl group or multiple methyl groups to increase the effects of modification.
6) N-acetylation
N-acetylation, or the transfer of an acetyl group to nitrogen, occurs in almost all eukaryotic proteins through both irreversible and reversible mechanisms. N-terminal acetylation requires the cleavage of the N-terminal methionine by methionine aminopeptidase (MAP) before replacing the amino acid with an acetyl group from acetyl-CoA by N-acetyltransferase (NAT) enzymes. This type of acetylation is co-translational, in that N-terminus is acetylated on growing polypeptide chains that are still attached to the ribosome. While 80-90% of eukaryotic proteins are acetylated in this manner, the exact biological significance is still unclear.
Acetylation at the ε-NH2 of lysine (termed lysine acetylation) on histone N-termini is a common method of regulating gene transcription. Histone acetylation is a reversible event that reduces chromosomal condensation to promote transcription, and the acetylation of these lysine residues is regulated by transcription factors that contain histone acetyletransferase (HAT) activity. While transcription factors with HAT activity act as transcription co-activators, histone deacetylase (HDAC) enzymes are co-repressors that reverse the effects of acetylation by reducing the level of lysine acetylation and increasing chromosomal condensation.
Sirtuins (silent information regulator) are a group of NAD-dependent deacetylases that target histones. As their name implies, they maintain gene silencing by hypoacetylating histones and have been reported to aid in maintaining genomic stability.
While acetylation was first detected in histones, cytoplasmic proteins have been reported to also be acetylated, and therefore acetylation seems to play a greater role in cell biology than simply transcriptional regulation. Furthermore, crosstalk between acetylation and other post-translational modifications, including phosphorylation, ubiquitination and methylation, can modify the biological function of the acetylated protein.
Protein acetylation can be detected by chromosome immunoprecipitation (ChIP) using acetyllysine-specific antibodies or by mass spectrometry, where an increase in histone by 42 mass units represents a single acetylation.
7) Lipidation
Lipidation is a method to target proteins to membranes in organelles (endoplasmic reticulum [ER], Golgi apparatus, and mitochondria), vesicles (endosomes, lysosomes) and the plasma membrane. The four types of lipidation are:
i. C-terminal glycosyl phosphatidylinositol (GPI) anchor
ii. N-terminal myristoylation
iii. S-myristoylation
iv. S-prenylation
Each type of modification gives proteins distinct membrane affinities, although all types of lipidation increase the hydrophobicity of a protein and thus its affinity for membranes. The different types of lipidation are also not mutually exclusive, in that two or more lipids can be attached to a given protein.
a) GPI anchors
GPI anchors tether cell surface proteins to the plasma membrane. These hydrophobic moieties are prepared in the ER, where they are then added to the nascent protein en bloc. GPI-anchored proteins are often localized to cholesterol- and sphingolipid-rich lipid rafts, which act as signaling platforms on the plasma membrane. This type of modification is reversible, as the GPI anchor can be released from the protein by phosphoinositol-specific phospholipase C. Indeed, this lipase is used in the detection of GPI-anchored proteins to release GPI-anchored proteins from membranes for gel separation and analysis by mass spectrometry.
b) N-myristoylation
N-myristoylation is a method to give proteins a hydrophobic handle for membrane localization. The myristoyl group is a 14-carbon saturated fatty acid (C14), which gives the protein sufficient hydrophobicity and affinity for membranes, but not enough to permanently anchor the protein in the membrane. N-myristoylation can therefore act as a conformational localization switch, in which protein conformational changes influence the availability of the handle for membrane attachment. Because of this conditional localization, signal proteins that selectively localize to membrane, such as Src-family kinases, are N-myristoylated.
N-myristoylation is facilitated specifically by N-myristoyltransferase (NMT) and uses myristoyl-CoA as the substrate to attach the myristoyl group to the N-terminal glycine. Because methionine is the N-terminal amino acid of all eukaryotic proteins, this PTM requires methionine cleavage by the above-mentioned MAP prior to addition of the myristoyl group; this represents one example of multiple PTMs on a single protein.
c) S-palmitoylation
S-palmitoylation adds a C16 palmitoyl group from palmitoyl-CoA to the thiolate side chain of cysteine residues via palmitoyl acyl transferases (PATs). Because of the longer hydrophobic group, this anchor can permanently anchor the protein to the membrane. This localization can be reversed, though, by thioesterases that break the link between the protein and the anchor; thus, S-palmitoylation is used as an on/off switch to regulate membrane localization. S-palmitoylation is often used to strengthen other types of lipidation, such as myristoylation or farnesylation (see below). S-palmitoylated proteins also selectively concentrate at lipid rafts.
d) S-prenylation
S-prenylation covalently adds a farnesyl (C15) or geranylgeranyl (C20) group to specific cysteine residues within 5 amino acids from the C-terminus via farnesyl transferase (FT) or geranylgeranyl transferases (GGT I and II). Unlike S-palmitoylation, S-prenylation is hydrolytically stable. Approximately 2% of all proteins are prenylated, including all members of the Ras superfamily. This group of molecular switches is farnesylated, geranylgeranylated or a combination of both. Additionally, these proteins have specific 4-amino acid motifs at the C-terminus that determine the type of prenylation at single or dual cysteines. Prenylation occurs in the ER and is often part of a stepwise process of PTMs that is followed by proteolytic cleavage by Rce1 and methylation by isoprenyl cysteine methyltransferase (ICMT).
8) Proteolysis
Peptide bonds are indefinitely stable under physiological conditions, and therefore cells require some mechanism to break these bonds. Proteases comprise a family of enzymes that cleave the peptide bonds of proteins and are critical in antigen processing, apoptosis, surface protein shedding and cell signaling.
The family of over 11,000 proteases varies in substrate specificity, mechanism of peptide cleavage, location in the cell and the length of activity. While this variation suggests a wide array of functionalities, proteases can generally be separated into groups based on the type of proteolysis. Degradative proteolysis is critical to remove unassembled protein subunits and misfolded proteins and to maintain protein concentrations at homeostatic concentrations by reducing a given protein to the level of small peptides and single amino acids. Proteases also play a biosynthetic role in cell biology that includes cleaving signal peptides from nascent proteins and activating zymogens, which are inactive enzyme precursors that require cleavage at specific sites for enzyme function. In this respect, proteases act as molecular switches to regulate enzyme activity.
Proteolysis is a thermodynamically favorable and irreversible reaction. Therefore, protease activity is tightly regulated to avoid uncontrolled proteolysis through temporal and/or spatial control mechanisms including regulation by cleavage in cis or trans and compartmentalization (e.g., proteasomes, lysosomes).
The diverse family of proteases can be classified by the site of action, such as aminopeptidases and carboxypeptidase, which cleave at the amino or carboxy terminus of a protein, respectively. Another type of classification is based on the active site groups of a given protease that are involved in proteolysis. Based on this classification strategy, greater than 90% of known proteases fall into one of four categories as follows:
a) Serine proteases
b) Cysteine proteases
c) Aspartic acid proteases
d) Zinc metalloproteases
4. Instrument:
LTQ-FT Mass Spectrometer
5. Data Analysis
Protein modifications that result in mass increases or losses to proteins are readily detectable by mass spectrometry. Creative Proteomics has extensive experience with characterizing acetylation, methylation, and phosphorylation modifications. Since there are over 300 known protein modifications, Creative Proteomics should be consulted during the design-phase of experiments in order to develop the appropriate strategy for mass spectrometric analysis of the modification. PTM analysis often requires purified protein in the amount of approximately 500 nanograms to 1 microgram.
6. Sample Preparation
Sample preparation involves cutting bands from SDS-PAGE gels, proteolytic digestion, and sample desalting. Investigators can take their samples through the complete preparation, or the proteomics staff can assist with some steps. If you choose to prepare your own gel samples, see our Tips for SDS-PAGE Gel Handling
7. SDS-PAGE Gel Handling Tips
1) Contamination of samples with keratin proteins is a constant battle in mass spectrometry labs. To help minimize this problem, gloves and a lab coat should be worn at all times when working with gels and their associated reagents.
2) Virtually any SDS-PAGE gel should be compatible with down-stream protein identification. Pre-cast gels are suggested to help reduce keratin contamination. Pre-cast gels from Invitrogen and BioRad have been used routinely at the Hutch.
3) Fresh staining reagents should be used (contaminants build up in re-used Coomassie stain). Silver staining and Coomassie staining are both compatible with mass spectrometry-based protein identification. Note: silver staining kits should not contain gluteraldehyde as a fixing agent. Most commercial kits will state if they are MS friendly.
4) After staining, gels should be washed thoroughly in ddH2O prior to band excision. Two washes at 15 minutes each should be sufficient. Longer washes are suggested for gels 1.5 mm and thicker.
5) Bands of interest should be cut from the gel with a clean, sharp razor blade. The band should be cut directly on the edge of the staining region; no boarders of clear acrylamide should be left around the band.
6) Cut bands should be place in Eppendorf tubes (other brands of tubes sometimes have contaminants that interfere with the mass spectrometry). We suggest a new box of tubes be opened and individual tubes be carefully selected, again to cut down on keratin contamination. We discourage tubes being used from "community" containers.
7) The Eppendorf tubes can be stored at room temperature or 4 °C until they are prepared. Storing the bands at -20 °C or -80 °C is discouraged as water in the gel bands will expand and cause the gel pieces to disintegrate, making the in-gel digestion procedure more difficult and reducing recovery of digested protein.
8) A blank region of the gel, approximately the size of the gel band of interest, should be submitted with the sample(s). This will act as a blank control.
9) Label the tubes well! (initials, date and sample name)
10) Tubes can be shipped via FedEx next day air.
8. FAQ
1) What are the concerns about mapping a phosphorylation site in my protein? (It is a large protein ~1000 aa.)
You will have two major concerns:
a) Coverage:
Can the mass spectrometry technique(s) deliver high sequence coverage? With higher coverage, there is a greater statistical chance to detect and identify the peptide containing a modification group.
b) Occupancy of the modification site:
What percent of the protein preparation is modified? If it is low, then the chance of detecting the modified peptide decreases. If the occupancy is high (>30%), then this works in the favor of identifying a modification site.
2) How can coverage be increased for mapping phosphorylation or methylation sites? (I understand that high coverage is essential for increasing the chance of mapping a PTM site.)
First, note that, due to its unique sequences, every protein has a different potential to be mapped at high sequence coverage.
Coverage for all proteins increases by:
a) Starting with a higher amount of protein (>5 μg of protein is desirable) for MS analysis
b) Making sure that the protein is pure
c) Using multiple MS instruments/techniques for analysis will increase coverage
d) Enriching the phosphopeptides using a TiO2 column
3) Will glycosylation sites inhibit the identification of my protein? (I think that my protein is glycosylated.)
If purity and concentration of the protein is high, then in most cases MS-based protein identification is still favorable.
Attached carbohydrate (CHO) chains, however, can impair MS-based identification of specific peptides through the following mechanism:
a) CHO chains can block trypsin cleavage
b) The variable mass of attached CHO chains can reduce glycopeptide detection.
c) Options: Cleave N-linked CHO chains with N-glycanase (PNGaseF) prior to gel loading and MS analysis
4) I want a preliminary analysis for potential phosphorylation sites in my protein. What is your lowest cost service? What are the limitations? What are your suggestions?
A lowest-cost preliminary analysis is a method which uses MALDI-TOF/TOF on a pure protein from a gel or a highly pure preparation. Limitations: The coverage of the protein will be low, if the protein is very large, or if it is impure, or if it is in low yield (<500 ng).
Suggestions: For a preliminary, low cost analysis, provide only a high quality sample (this would be a single band on a gel that is in the 1 μg range).
9. References
1) International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature. 431, 931-45.
2) Jensen O. N. (2004) Modification-specific proteomics: Characterization of post-translational modifications by mass spectrometry. Curr Opin Chem Biol. 8, 33-41.
3) Ayoubi T. A. and Van De Ven W. J. (1996) Regulation of gene expression by alternative promoters. FASEB J. 10, 453-60.
4) Walsh C. (2006) Posttranslational modification of proteins: Expanding nature's inventory. Englewood, Colo.: Roberts and Co. Publishers. xxi, 490 p. p.
5) Gaston B. M. et al. (2003) S-nitrosylation signaling in cell biology. Mol Interv. 3, 253-63.
6) Jaffrey S. R. and Snyder S. H. (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE. 2001, pl1.
7) Han P. and Chen C. (2008) Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of S-nitrosylated proteins. Rapid Commun Mass Spectrum. 22, 1137-45.
8) Imai S. et al. (2000) Transcriptional silencing and longevity protein SIR2 is an NAD-dependent histone deacetylase. Nature. 403, 795-800.
9) Glozak M. A. et al. (2005) Acetylation and deacetylation of non-histone proteins. Gene. 363, 15-23.
10) Yang X. J. and Seto E. (2008) Lysine acetylation: Codified crosstalk with other posttranslational modifications. Mol Cell. 31, 449-61.
11) Grotenbreg, G., Ploegh, H. (2007) Chemical biology: dressed-up proteins. Nature, 446:993-995.
12) Geiss-Friedlander, R., Melchior, F. (2007) Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol, 8:947-956.
13) Morrison, R.S., Kinoshita, Y., Johnson M.D., Uo, T., Ho, J.T., BcBee, J.K., Conrads, T.P., Veenstra, T.D. (2002) Proteomic Analysis in the Neurosciences. Mol Cell Proteomics, 1:553–560.
14) Hubbard, M.J., Cohen, P. (1993) On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem. Sci, 18:172-177.
15) Pawson, T., Scott, J.D. (1997) Signaling through scaffold, anchoring, and adaptor proteins. Science, 278:2075-80.
16) Hunter, T. (2000). "Signaling - 2000 and beyond." Cell 100(1):113-127.
17) Cohen, P.T.W. (2002). Protein phosphatase 1- targeted in many directions. J Cell Sci, 115:241-256.
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