Protein mass spectrometry refers to the application of
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
to the study of
proteins
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, respo ...
. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their
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 ribosome ...
s, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in
proteomics
Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...
. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids.
The two primary methods used for the ionization of protein in mass spectrometry are
electrospray ionization
Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules becaus ...
(ESI) and
matrix-assisted laser desorption/ionization
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 ...
(MALDI). These ionization techniques are used in conjunction with mass analyzers such as
tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A comm ...
. In general, the proteins are analyzed either in a "
top-down
Top-down may refer to:
Arts and entertainment
* " Top Down", a 2007 song by Swizz Beatz
* "Top Down", a song by Lil Yachty from ''Lil Boat 3''
* "Top Down", a song by Fifth Harmony from ''Reflection'' Science
* Top-down reading, is a part of ...
" approach in which proteins are analyzed intact, or a "
bottom-up" approach in which protein are first digested into fragments. An intermediate "middle-down" approach in which larger peptide fragments are analyzed may also sometimes be used.
History
The application of mass spectrometry to study proteins became popularized in the 1980s after the development of
MALDI
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 ...
and
ESI. These ionization techniques have played a significant role in the characterization of proteins. (MALDI) Matrix-assisted laser desorption ionization was coined in the late 80's by
Franz Hillenkamp
Franz Hillenkamp (March 18, 1936 – August 22, 2014) was a German scientist known for his development of the laser microprobe mass analyzer and, with Michael Karas, matrix-assisted laser desorption/ionization (MALDI).
Early life and education
Fr ...
and
Michael Karas.
Hillenkamp, Karas and their fellow researchers were able to ionize the amino acid
alanine
Alanine (symbol Ala or A), or α-alanine, is an α-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side c ...
by mixing it with the amino acid
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 ...
and irradiated with a pulse 266 nm laser.
Though important, the breakthrough did not come until 1987. In 1987,
Koichi Tanaka
is a Japanese electrical engineer who shared the Nobel Prize in Chemistry in 2002 for developing a novel method for mass spectrometric analyses of biological macromolecules with John Bennett Fenn and Kurt Wüthrich (the latter for work in N ...
used the "ultra fine metal plus liquid matrix method" and ionized biomolecules the size of 34,472 Da protein carboxypeptidase-A.
In 1968,
Malcolm Dole
Malcolm Dole (March 4, 1903 – November 29, 1990) was an American chemist known for the Dole Effect in which he proved that the atomic weight of oxygen in air is greater than that of oxygen in water and for his work on electrospray ionizati ...
reported the first use of electrospray ionization with mass spectrometry. Around the same time MALDI became popularized,
John Bennett Fenn
John Bennett Fenn (June 15, 1917December 10, 2010) was an American professor of analytical chemistry who was awarded a share of the Nobel Prize in Chemistry in 2002. Fenn shared half of the award with Koichi Tanaka for their work in mass spectro ...
was cited for the development of electrospray ionization.
Koichi Tanaka received the 2002
Nobel Prize in Chemistry
)
, image = Nobel Prize.png
, alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
alongside John Fenn, and
Kurt Wüthrich
Kurt Wüthrich (born 4 October 1938 in Aarberg, Canton of Bern) is a Swiss chemist/biophysicist and Nobel Chemistry laureate, known for developing nuclear magnetic resonance (NMR) methods for studying biological macromolecules.
Education and e ...
"for the development of methods for identification and structure analyses of biological macromolecules."
These ionization methods have greatly facilitated the study of proteins by mass spectrometry. Consequently, protein mass spectrometry now plays a leading role in protein characterization.
Methods and approaches
Techniques
Mass spectrometry of proteins requires that the proteins in solution or solid state be turned into an ionized form in the gas phase before they are injected and accelerated in an electric or magnetic field for analysis. The two primary methods for ionization of proteins are
electrospray ionization
Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules becaus ...
(ESI) and
matrix-assisted laser desorption/ionization
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 ...
(MALDI). In electrospray, the ions are created from proteins in solution, and it allows fragile molecules to be ionized intact, sometimes preserving non-covalent interactions. In MALDI, the proteins are embedded within a matrix normally in a solid form, and ions are created by pulses of laser light. Electrospray produces more multiply-charged ions than MALDI, allowing for measurement of high mass protein and better fragmentation for identification, while MALDI is fast and less likely to be affected by contaminants, buffers and additives.
Whole-protein mass analysis is primarily conducted using either
time-of-flight
Time of flight (ToF) is the measurement of the time taken by an object, particle or wave (be it acoustic, electromagnetic, etc.) to travel a distance through a medium. This information can then be used to measure velocity or path length, or as a w ...
(TOF) MS, or
Fourier transform ion cyclotron resonance
Fourier-transform ion cyclotron resonance mass spectrometry is a type of mass analyzer (or mass spectrometer) for determining the mass-to-charge ratio (''m''/''z'') of ions based on the ion cyclotron resonance, cyclotron frequency of the ions in a ...
(FT-ICR). These two types of instrument are preferable here because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. Electrospray ionization of a protein often results in generation of multiple charged species of 800 <
''m/z'' < 2000 and the resultant spectrum can be deconvoluted to determine the protein's average mass to within 50 ppm or better using TOF or
ion-trap instruments.
Mass analysis of proteolytic peptides is a popular method of protein characterization, as cheaper instrument designs can be used for characterization. Additionally, sample preparation is easier once whole proteins have been digested into smaller peptide fragments. The most widely used instrument for peptide mass analysis are the MALDI-TOF instruments as they permit the acquisition of
peptide mass fingerprints (PMFs) at high pace (1 PMF can be analyzed in approx. 10 sec). Multiple stage quadrupole-time-of-flight and the
quadrupole ion trap
A quadrupole ion trap or paul trap is a type of ion trap that uses dynamic electric fields to trap charged particles. They are also called radio frequency (RF) traps or Paul traps in honor of Wolfgang Paul, who invented the device and shared the ...
also find use in this application.
Tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A comm ...
(MS/MS) is used to measure fragmentation spectra and identify proteins at high speed and accuracy. Collision-induced dissociation is used in mainstream applications to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of this simplicity in fragmentation, it is possible to use the observed fragment masses to match with a database of predicted masses for one of many given peptide sequences. Tandem MS of whole protein ions has been investigated recently using
electron capture dissociation
Electron-capture dissociation (ECD) is a method of fragmenting gas-phase ions for structure elucidation of peptides and proteins in tandem mass spectrometry. It is one of the most widely used techniques for activation and dissociation of mass sel ...
and has demonstrated extensive sequence information in principle but is not in common practice.
Approaches
In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as "
top-down
Top-down may refer to:
Arts and entertainment
* " Top Down", a 2007 song by Swizz Beatz
* "Top Down", a song by Lil Yachty from ''Lil Boat 3''
* "Top Down", a song by Fifth Harmony from ''Reflection'' Science
* Top-down reading, is a part of ...
" strategy of protein analysis as it involves starting with the whole mass and then pulling it apart. The top-down approach however is mostly limited to low-throughput single-protein studies due to issues involved in handling whole proteins, their heterogeneity and the complexity of their analyses.
In the second approach, referred to as the "
bottom-up" MS, proteins are enzymatically digested into smaller
peptides
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.
A p ...
using a
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 ...
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 dig ...
. Subsequently, these peptides are introduced into the mass spectrometer and identified by
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 ...
or
tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A comm ...
. Hence, this approach uses identification at the peptide level to infer the existence of proteins pieced back together with ''de novo'' repeat detection.
The smaller and more uniform fragments are easier to analyze than intact proteins and can be also determined with high accuracy, this "bottom-up" approach is therefore the preferred method of studies in proteomics. A further approach that is beginning to be useful is the intermediate "middle-down" approach in which proteolytic peptides larger than the typical tryptic peptides are analyzed.
Protein and peptide fractionation
![Mass spectrometry protocol](https://upload.wikimedia.org/wikipedia/commons/1/1f/Mass_spectrometry_protocol.png)
Proteins of interest are usually part of a complex mixture of multiple proteins and molecules, which co-exist in the biological medium. This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal amounts of material, while in biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized using
electrospray
The name electrospray is used for an apparatus that employs electricity to disperse a liquid or for the fine aerosol resulting from this process. High voltage is applied to a liquid supplied through an emitter (usually a glass or metallic capilla ...
or
MALDI
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 ...
, the more abundant species have a tendency to "drown" or suppress signals from less abundant ones. Second, mass spectrum from a complex mixture is very difficult to interpret due to the overwhelming number of mixture components. This is exacerbated by the fact that enzymatic digestion of a protein gives rise to a large number of peptide products.
In light of these problems, the methods of one- and two-dimensional gel electrophoresis and high performance liquid chromatography are widely used for separation of proteins. The first method fractionates whole proteins via
two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first ...
. The first-dimension of 2D gel is
isoelectric focusing
Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis usually performed on proteins in a gel that takes ad ...
(IEF). In this dimension, the protein is separated by its isoelectric point (pI) and the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This dimension separates the protein according to its molecular weight. Once this step is completed in-gel digestion occurs. In some situations, it may be necessary to combine both of these techniques. Gel spots identified on a 2D Gel are usually attributable to one protein. If the identity of the protein is desired, usually the method of
in-gel digestion The in-gel digestion step is a part of the sample preparation for the mass spectrometric identification of proteins in course of proteomic analysis. The method was introduced in 1992 by Rosenfeld.Rosenfeld, J et al., ''Anal Biochem'', 1992, 203 ( ...
is applied, where the protein spot of interest is excised, and digested proteolytically. The peptide masses resulting from the digestion can be determined by mass spectrometry using
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 ...
. If this information does not allow unequivocal identification of the protein, its peptides can be subject to
tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A comm ...
for
''de novo'' sequencing. Small changes in mass and charge can be detected with 2D-PAGE. The disadvantages with this technique are its small dynamic range compared to other methods, some proteins are still difficult to separate due to their acidity, basicity, hydrophobicity, and size (too large or too small).
The second method,
high performance liquid chromatography
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pa ...
is used to fractionate peptides after enzymatic digestion. Characterization of protein mixtures using HPLC/MS is also called shotgun proteomics and MuDPIT (Multi-Dimensional Protein Identification Technology). A peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography. The eluent from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.
Applications
Protein identification
There are two main ways MS is used to identify proteins.
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 ...
uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample. Purification steps therefore limit the throughput of the peptide mass fingerprinting approach. Alternatively, peptides can be fragmented with MS/MS to more definitively identify them.
MS is also the preferred method for the identification of
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 ribosome ...
s in proteins versus other approaches such as antibody-based methods.
''De novo'' (peptide) sequencing
''De novo'' peptide sequencing for mass spectrometry is typically performed without prior knowledge of the amino acid sequence. It is the process of assigning amino acids from
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.
A ...
fragment
mass
Mass is an intrinsic property of a body. It was traditionally believed to be related to the quantity of matter in a physical body, until the discovery of the atom and particle physics. It was found that different atoms and different elementar ...
es 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, respo ...
. ''De novo'' sequencing has proven successful for confirming and expanding upon results from database searches.
As ''de novo'' sequencing is based on mass and some amino acids have identical masses (e.g.
leucine
Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α-amino group (which is in the protonated −NH3+ form under biological conditions), an α- ca ...
and
isoleucine
Isoleucine (symbol Ile or I) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the deprot ...
), accurate manual sequencing can be difficult. Therefore, it may be necessary to utilize a sequence homology search application to work in tandem between a database search and ''de novo'' sequencing to address this inherent limitation.
Database searching has the advantage of quickly identifying sequences, provided they have already been documented in a database. Other inherent limitations of database searching include sequence modifications/mutations (some database searches do not adequately account for alterations to the 'documented' sequence, thus can miss valuable information), the unknown (if a sequence is not documented, it will not be found), false positives, and incomplete and corrupted data.
An annotated
peptide spectral library can also be used as a reference for protein/peptide identification. It offers the unique strength of reduced search space and increased specificity. The limitations include spectra not included in the library will not be identified, spectra collected from different types of mass spectrometers can have quite distinct features, and reference spectra in the library may contain noise peaks, which may lead to false positive identifications. A number of different algorithmic approaches have been described to identify peptides and proteins from
tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A comm ...
(MS/MS), peptide ''de novo'' sequencing and sequence tag-based searching.
Antigen presentation
Antigen presentation is the first step in educating the immune system to recognize new pathogens. To this end, antigen presenting cells expose protein fragments via
MHC molecules to the immune system. Not all protein fragments bind, however, to the MHC molecules of a certain individual. Using mass spectrometry, the true spectrum of molecules presented to the immune system can be determined.
Protein quantitation
Multiple methods allow for the quantitation of proteins by mass spectrometry, and recent advances have enabled quantifying thousands of proteins in single cells. Typically, stable (e.g. non-radioactive) heavier
isotope
Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers (mass numbers) ...
s of
carbon
Carbon () is a chemical element with the symbol C and atomic number 6. It is nonmetallic and tetravalent
In chemistry, the valence (US spelling) or valency (British spelling) of an element is the measure of its combining capacity with o ...
(
13C) or
nitrogen
Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
(
15N) are incorporated into one sample while the other one is labeled with corresponding light isotopes (e.g.
12C and
14N).
The two samples are mixed before the analysis. Peptides derived from the different samples can be distinguished due to their mass difference. The ratio of their peak intensities corresponds to the relative abundance ratio of the peptides (and proteins). The most popular methods for isotope labeling are
SILAC Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitativ ...
(stable isotope labeling by amino acids in cell culture), trypsin-catalyzed
18O labeling,
ICAT (isotope coded affinity tagging),
iTRAQ (isobaric tags for relative and absolute quantitation).
“Semi-quantitative” mass spectrometry can be performed without labeling of samples.
Typically, this is done with MALDI analysis (in linear mode). The peak intensity, or the peak area, from individual molecules (typically proteins) is here correlated to the amount of protein in the sample. However, the individual signal depends on the primary structure of the protein, on the complexity of the sample, and on the settings of the instrument. Other types of "label-free" quantitative mass spectrometry, uses the spectral counts (or peptide counts) of digested proteins as a means for determining relative protein amounts.
Protein structure determination
Characteristics indicative of the
3-dimensional structure of proteins can be probed with mass spectrometry in various ways. By using chemical crosslinking to couple parts of the protein that are close in space, but far apart in sequence, information about the overall structure can be inferred. By following the
exchange of amide protons with
deuterium
Deuterium (or hydrogen-2, symbol or deuterium, also known as heavy hydrogen) is one of two Stable isotope ratio, stable isotopes of hydrogen (the other being Hydrogen atom, protium, or hydrogen-1). The atomic nucleus, nucleus of a deuterium ato ...
from the solvent, it is possible to probe the solvent accessibility of various parts of the protein.
Hydrogen-deuterium exchange mass spectrometry has been used to study proteins and their conformations for over 20 years. This type of protein structural analysis can be suitable for proteins that are challenging for other structural methods. Another interesting avenue in protein structural studies is laser-induced covalent labeling. In this technique, solvent-exposed sites of the protein are modified by hydroxyl radicals. Its combination with rapid mixing has been used in protein folding studies.
Proteogenomics
In what is now commonly referred to as
proteogenomics
Proteogenomics is a field of biological research that utilizes a combination of proteomics, genomics, and transcriptomics to aid in the discovery and identification of peptides. Proteogenomics is used to identify new peptides by comparing MS/MS sp ...
, peptides identified with
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
are used for improving gene annotations (for example, gene start sites) and protein annotations. Parallel analysis of the genome and the proteome facilitates discovery of post-translational modifications and proteolytic events,
especially when comparing multiple species.
References
{{DEFAULTSORT:Protein Mass Spectrometry
Mass spectrometry
Proteomics