Protein sequencing is the practical process of determining the
amino acid sequence
Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynthe ...
of all or part of a
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
or
peptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
...
. This may serve to identify the protein or characterize its
post-translational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribos ...
s. Typically, partial sequencing of a protein provides sufficient information (one or more sequence tags) to identify it with reference to databases of protein sequences derived from the conceptual
translation
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...
of
gene
In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
s.
The two major direct methods of protein sequencing are
mass spectrometry and
Edman degradation
Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residu ...
using a
protein sequenator (sequencer). Mass spectrometry methods are now the most widely used for protein sequencing and identification but Edman degradation remains a valuable tool for characterizing a protein's
''N''-terminus.
Determining amino acid composition
It is often desirable to know the unordered amino acid composition of a protein prior to attempting to find the ordered sequence, as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to distinguish between ambiguous results. Knowledge of the frequency of certain amino acids may also be used to choose which
protease
A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the ...
to use for digestion of the protein. The misincorporation of low levels of non-standard amino acids (e.g. norleucine) into proteins may also be determined. A generalized method often referred to as ''amino acid analysis''
for determining amino acid frequency is as follows:
#Hydrolyse a known quantity of protein into its constituent amino acids.
#Separate and quantify the amino acids in some way.
Hydrolysis
Hydrolysis
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution, elimination, and solvation reactions in which water is the nucleophile.
Biological hydrolys ...
is done by heating a sample of the protein in 6 M
hydrochloric acid
Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid
Acid strength is the tendency of an acid, symbol ...
to 100–110 °C for 24 hours or longer. Proteins with many bulky
hydrophobic
In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water.
Hydrophobic molecules tend to be nonpolar and, t ...
groups may require longer heating periods. However, these conditions are so vigorous that some amino acids (
serine,
threonine,
tyrosine
-Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...
,
tryptophan
Tryptophan (symbol Trp or W)
is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...
,
glutamine
Glutamine (symbol Gln or Q) is an α-amino acid that is used in the biosynthesis of proteins. Its side chain is similar to that of glutamic acid, except the carboxylic acid group is replaced by an amide. It is classified as a charge-neutral ...
, and
cysteine) are degraded. To circumvent this problem, Biochemistry Online suggests heating separate samples for different times, analysing each resulting solution, and extrapolating back to zero hydrolysis time. Rastall suggests a variety of reagents to prevent or reduce degradation, such as
thiol
In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl gro ...
reagents or
phenol
Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it ...
to protect tryptophan and tyrosine from attack by chlorine, and pre-oxidising cysteine. He also suggests measuring the quantity of
ammonia
Ammonia is an inorganic compound of nitrogen and hydrogen with the formula . A stable binary hydride, and the simplest pnictogen hydride, ammonia is a colourless gas with a distinct pungent smell. Biologically, it is a common nitrogenous wa ...
evolved to determine the extent of
amide hydrolysis.
Separation and quantitation
The amino acids can be separated by
ion-exchange chromatography
Ion chromatography (or ion-exchange chromatography) separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino a ...
then derivatized to facilitate their detection. More commonly, the amino acids are derivatized then resolved by
reversed phase HPLC.
An example of the ion-exchange chromatography is given by the NTRC using sulfonated polystyrene as a matrix, adding the amino acids in acid solution and passing a buffer of steadily increasing
pH through the column. Amino acids are eluted when the pH reaches their respective
isoelectric point
The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net electrical charge or is electrically neutral in the statistical mean. The standard nomenclature to represent the isoelectric point is pH(I). However, pI is also u ...
s. Once the amino acids have been separated, their respective quantities are determined by adding a reagent that will form a coloured derivative. If the amounts of amino acids are in excess of 10 nmol,
ninhydrin
Ninhydrin (2,2-dihydroxyindane-1,3-dione) is an organic compound with the formula C6H4(CO)2C(OH)2. It is used to detect ammonia and amines. Upon reaction with these amines, ninhydrin gets converted into deep blue or purple derivatives, which are ...
can be used for this; it gives a yellow colour when reacted with proline, and a vivid purple with other amino acids. The concentration of amino acid is proportional to the absorbance of the resulting solution. With very small quantities, down to 10 pmol, fluorescent derivatives can be formed using reagents such as
ortho-phthaldehyde (OPA) or
fluorescamine.
Pre-column derivatization may use the Edman reagent to produce a derivative that is detected by UV light. Greater sensitivity is achieved using a reagent that generates a fluorescent derivative. The derivatized amino acids are subjected to reversed phase chromatography, typically using a C8 or C18
silica column and an optimised
elution
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.
In a liquid chromatography experiment, for exam ...
gradient. The eluting amino acids are detected using a UV or fluorescence detector and the peak areas compared with those for derivatised standards in order to quantify each amino acid in the sample.
''N''-terminal amino acid analysis
Determining which amino acid forms the
''N''-terminus of a
peptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
...
chain is useful for two reasons: to aid the ordering of individual peptide fragments' sequences into a whole chain, and because the first round of
Edman degradation
Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residu ...
is often contaminated by impurities and therefore does not give an accurate determination of the ''N''-terminal amino acid. A generalised method for ''N''-terminal amino acid analysis follows:
#React the peptide with a reagent that will selectively label the terminal amino acid.
#Hydrolyse the protein.
#Determine the amino acid by chromatography and comparison with standards.
There are many different reagents which can be used to label terminal amino acids. They all react with amine groups and will therefore also bind to amine groups in the side chains of amino acids such as lysine - for this reason it is necessary to be careful in interpreting chromatograms to ensure that the right spot is chosen. Two of the more common reagents are Sanger's reagent (
1-fluoro-2,4-dinitrobenzene) and dansyl derivatives such as
dansyl chloride
Dansyl chloride or 5-(DimethylAmino)Naphthalene-1-SulfonYL chloride is a reagent that reacts with primary amino groups in both aliphatic and aromatic amines to produce stable blue- or blue-green–fluorescent sulfonamide adducts. It can also be m ...
.
Phenylisothiocyanate, the reagent for the Edman degradation, can also be used. The same questions apply here as in the determination of amino acid composition, with the exception that no stain is needed, as the reagents produce coloured derivatives and only qualitative analysis is required. So the amino acid does not have to be eluted from the chromatography column, just compared with a standard. Another consideration to take into account is that, since any amine groups will have reacted with the labelling reagent, ion exchange chromatography cannot be used, and
thin layer chromatography
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures.
Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a t ...
or
high-pressure liquid chromatography should be used instead.
C-terminal amino acid analysis
The number of methods available for
C-terminal
The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is ...
amino acid analysis is much smaller than the number of available methods of N-terminal analysis. The most common method is to add
carboxypeptidase
A carboxypeptidase ( EC number 3.4.16 - 3.4.18) is a protease enzyme that hydrolyzes (cleaves) a peptide bond at the carboxy-terminal (C-terminal) end of a protein or peptide. This is in contrast to an aminopeptidases, which cleave peptide bonds ...
s to a solution of the protein, take samples at regular intervals, and determine the terminal amino acid by analysing a plot of amino acid concentrations against time. This method will be very useful in the case of polypeptides and protein-blocked N termini. C-terminal sequencing would greatly help in verifying the primary structures of proteins predicted from DNA sequences and to detect any posttranslational processing of gene products from known codon sequences.
Edman degradation
The
Edman degradation
Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residu ...
is a very important reaction for protein sequencing, because it allows the ordered amino acid composition of a protein to be discovered. Automated Edman sequencers are now in widespread use, and are able to sequence peptides up to approximately 50 amino acids long. A reaction scheme for sequencing a protein by the Edman degradation follows; some of the steps are elaborated on subsequently.
#Break any
disulfide bridge
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
s in the protein with a
reducing agent
In chemistry, a reducing agent (also known as a reductant, reducer, or electron donor) is a chemical species that "donates" an electron to an (called the , , , or ).
Examples of substances that are commonly reducing agents include the Earth me ...
like
2-mercaptoethanol
2-Mercaptoethanol (also β-mercaptoethanol, BME, 2BME, 2-ME or β-met) is the chemical compound with the formula HOCH2CH2SH. ME or βME, as it is commonly abbreviated, is used to reduce disulfide bonds and can act as a biological antioxidant by s ...
. A
protecting group such as
iodoacetic acid
Iodoacetic acid is a derivative of acetic acid. It is a toxic compound, because, like many alkyl halides, it is an alkylating agent.
It reacts with cysteine residues in proteins. It is often used to modify SH-groups to prevent the re-formation ...
may be necessary to prevent the bonds from re-forming.
#Separate and purify the individual chains of the protein complex, if there are more than one.
#Determine the amino acid composition of each chain.
#Determine the terminal amino acids of each chain.
#Break each chain into fragments under 50 amino acids long.
#Separate and purify the fragments.
#Determine the sequence of each fragment.
#Repeat with a different pattern of cleavage.
#Construct the sequence of the overall protein.
Digestion into peptide fragments
Peptides longer than about 50–70 amino acids long cannot be sequenced reliably by the Edman degradation. Because of this, long protein chains need to be broken up into small fragments that can then be sequenced individually. Digestion is done either by
endopeptidase
Endopeptidase or endoproteinase are proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule), in contrast to exopeptidases, which break peptide bonds from end-pieces of terminal amino acids. For this ...
s such as
trypsin
Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the d ...
or
pepsin
Pepsin is an endopeptidase that breaks down proteins into smaller peptides. It is produced in the gastric chief cells of the stomach lining and is one of the main digestive enzymes in the digestive systems of humans and many other animals, w ...
or by chemical reagents such as
cyanogen bromide
Cyanogen bromide is the inorganic compound with the formula (CN)Br or BrCN. It is a colorless solid that is widely used to modify biopolymers, fragment proteins and peptides (cuts the C-terminus of methionine), and synthesize other compounds. ...
. Different enzymes give different cleavage patterns, and the overlap between fragments can be used to construct an overall sequence.
Reaction
The peptide to be sequenced is
adsorbed
Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the ''adsorbate'' on the surface of the ''adsorbent''. This process differs from absorption, in which a ...
onto a solid surface. One common
substrate is glass fibre coated with
polybrene
Hexadimethrine bromide (commercial brand name Polybrene) is a cationic polymer with several uses. Currently, it is primarily used to increase the efficiency of transduction of certain cells with retrovirus in cell culture. Hexadimethrine bromi ...
, a
cationic polymer. The Edman reagent,
phenylisothiocyanate (PITC), is added to the adsorbed peptide, together with a mildly basic
buffer solution
A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is ...
of 12%
trimethylamine
Trimethylamine (TMA) is an organic compound with the formula N(CH3)3. It is a colorless, hygroscopic, and flammable tertiary amine. It is a gas at room temperature but is usually sold as a 40% solution in water. (It is also sold in pressurized ...
. This reacts with the amine group of the N-terminal amino acid.
The terminal amino acid can then be selectively detached by the addition of
anhydrous acid. The derivative then
isomerises to give a substituted
phenylthiohydantoin, which can be washed off and identified by chromatography, and the cycle can be repeated. The efficiency of each step is about 98%, which allows about 50 amino acids to be reliably determined.
Protein sequencer
A protein sequenator is a machine that performs Edman degradation in an automated manner. A sample of the protein or peptide is immobilized in the reaction vessel of the protein sequenator and the Edman degradation is performed. Each cycle releases and derivatises one amino acid from the protein or peptide's ''N''-terminus and the released amino-acid derivative is then identified by HPLC. The sequencing process is done repetitively for the whole
polypeptide until the entire measurable sequence is established or for a pre-determined number of cycles.
Identification by mass spectrometry
Protein identification is the process of assigning a name to a protein of interest (POI), based on its amino-acid sequence. Typically, only part of the protein’s sequence needs to be determined experimentally in order to identify the protein with reference to databases of protein sequences deduced from the DNA sequences of their genes. Further protein characterization may include confirmation of the actual N- and C-termini of the POI, determination of sequence variants and identification of any post-translational modifications present.
Proteolytic digests
A general scheme for protein identification is described.
# The POI is isolated, typically by
SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...
or
chromatography
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system ( ...
.
# The isolated POI may be chemically modified to stabilise Cysteine residues (e.g. S-amidomethylation or S-carboxymethylation).
# The POI is digested with a specific protease to generate peptides.
Trypsin
Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the d ...
, which cleaves selectively on the C-terminal side of Lysine or Arginine residues, is the most commonly used protease. Its advantages include i) the frequency of Lys and Arg residues in proteins, ii) the high specificity of the enzyme, iii) the stability of the enzyme and iv) the suitability of tryptic peptides for mass spectrometry.
# The peptides may be desalted to remove ionizable contaminants and subjected to
MALDI-TOF
In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. It has been applied to the analysis of ...
mass spectrometry. Direct measurement of the masses of the peptides may provide sufficient information to identify the protein (see
Peptide mass fingerprinting
Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately ...
) but further fragmentation of the peptides inside the mass spectrometer is often used to gain information about the peptides’ sequences. Alternatively, peptides may be desalted and separated by
reversed phase HPLC and introduced into a mass spectrometer via an
ESI source. LC-ESI-MS may provide more information than MALDI-MS for protein identification but uses more instrument time.
# Depending on the type of mass spectrometer, fragmentation of peptide ions may occur via a variety of mechanisms such as
Collision-induced dissociation
Collision-induced dissociation (CID), also known as collisionally activated dissociation (CAD), is a mass spectrometry technique to induce fragmentation of selected ions in the gas phase. The selected ions (typically molecular ions or protonate ...
(CID) or
Post-source decay (PSD). In each case, the pattern of fragment ions of a peptide provides information about its sequence.
# Information including the measured mass of the putative peptide ions and those of their fragment ions is then matched against calculated mass values from the conceptual (in-silico) proteolysis and fragmentation of databases of protein sequences. A successful match will be found if its score exceeds a threshold based on the analysis parameters. Even if the actual protein is not represented in the database, error-tolerant matching allows for the putative identification of a protein based on similarity to
homologous proteins. A variety of software packages are available to perform this analysis.
# Software packages usually generate a report showing the identity (accession code) of each identified protein, its matching score, and provide a measure of the relative strength of the matching where multiple proteins are identified.
# A diagram of the matched peptides on the sequence of the identified protein is often used to show the sequence coverage (% of the protein detected as peptides). Where the POI is thought to be significantly smaller than the matched protein, the diagram may suggest whether the POI is an N- or C-terminal fragment of the identified protein.
De novo sequencing
The pattern of fragmentation of a peptide allows for direct determination of its sequence by
''de novo'' sequencing. This sequence may be used to match databases of protein sequences or to investigate
post-translational
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosom ...
or chemical modifications. It may provide additional evidence for protein identifications performed as above.
N- and C-termini
The peptides matched during protein identification do not necessarily include the N- or C-termini predicted for the matched protein. This may result from the N- or C-terminal peptides being difficult to identify by MS (e.g. being either too short or too long), being post-translationally modified (e.g. N-terminal acetylation) or genuinely differing from the prediction. Post-translational modifications or truncated termini may be identified by closer examination of the data (i.e. ''de novo'' sequencing). A repeat digest using a protease of different specificity may also be useful.
Post-translational modifications
Whilst detailed comparison of the MS data with predictions based on the known protein sequence may be used to define post-translational modifications, targeted approaches to data acquisition may also be used. For instance, specific enrichment of phosphopeptides may assist in identifying
phosphorylation sites in a protein. Alternative methods of peptide fragmentation in the mass spectrometer, such as
ETD or
ECD, may give complementary sequence information.
Whole-mass determination
The protein’s whole mass is the sum of the masses of its amino-acid residues plus the mass of a water molecule and adjusted for any post-translational modifications. Although proteins ionize less well than the peptides derived from them, a protein in solution may be able to be subjected to ESI-MS and its mass measured to an accuracy of 1 part in 20,000 or better. This is often sufficient to confirm the termini (thus that the protein’s measured mass matches that predicted from its sequence) and infer the presence or absence of many post-translational modifications.
Limitations
Proteolysis does not always yield a set of readily analyzable peptides covering the entire sequence of POI. The fragmentation of peptides in the mass spectrometer often does not yield ions corresponding to cleavage at each peptide bond. Thus, the deduced sequence for each peptide is not necessarily complete. The standard methods of fragmentation do not distinguish between leucine and isoleucine residues since they are isomeric.
Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminus has been chemically modified (e.g. by acetylation or formation of Pyroglutamic acid). Edman degradation is generally not useful to determine the positions of disulfide bridges. It also requires peptide amounts of 1 picomole or above for discernible results, making it less sensitive than
mass spectrometry.
Predicting from DNA/RNA sequences
In biology, proteins are produced by
translation
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...
of messenger RNA (mRNA) with the protein sequence deriving from the sequence of codons in the mRNA. The mRNA is itself formed by the
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
of genes and may be further modified. These processes are sufficiently understood to use computer algorithms to automate predictions of protein sequences from DNA sequences, such as from whole-genome DNA-sequencing projects, and have led to the generation of large databases of protein sequences such as
UniProt
UniProt is a freely accessible database of protein sequence and functional information, many entries being derived from genome sequencing projects. It contains a large amount of information about the biological function of proteins derived from ...
. Predicted protein sequences are an important resource for protein identification by mass spectrometry.
Historically, short protein sequences (10 to 15 residues) determined by Edman degradation were back-translated into DNA sequences that could be used as probes or primers to isolate
molecular clones of the corresponding gene or complementary DNA. The sequence of the cloned DNA was then determined and used to deduce the full amino-acid sequence of the protein.
Bioinformatics tools
Bioinformatics tools exist to assist with interpretation of mass spectra (see
De novo peptide sequencing In mass spectrometry, de novo peptide sequencing is the method in which a peptide amino acid sequence is determined from tandem mass spectrometry.
Knowing the amino acid sequence of peptides from a protein digest is essential for studying the biolo ...
), to compare or analyze protein sequences (see
Sequence analysis
In bioinformatics, sequence analysis is the process of subjecting a DNA, RNA or peptide sequence to any of a wide range of analytical methods to understand its features, function, structure, or evolution. Methodologies used include sequence alig ...
), or search databases using peptide or protein sequences (see
BLAST
Blast or The Blast may refer to:
*Explosion, a rapid increase in volume and release of energy in an extreme manner
*Detonation, an exothermic front accelerating through a medium that eventually drives a shock front
Film
* ''Blast'' (1997 film), ...
).
Applications to cryptography
The difficulty of protein sequencing was recentl
proposedas a basis for creating k-time programs, programs that run exactly k times before self-destructing. Such a thing is impossible to build purely in software because all software is inherently clonable an unlimited number of times.
See also
*
Proteomics
*
DNA sequencing
*
Klaus Biemann
*
Donald F. Hunt
Donald F. Hunt is the University Professor of Chemistry and Pathology at the University of Virginia. He is known for his research in the field of mass spectrometry, he developed electron capture negative ion mass spectrometry. He has received mult ...
*
Matthias Mann
*
John R. Yates
References
Further reading
*
{{Authority control
Cell biology
Proteomic sequencing