Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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Term Definition

General

Analysis of variance

(ANOVA)

Statistical tool to test differences among several

group means, whether they are different enough

not to have occurred by chance.

Background subtraction The process in which the background (chemical

and detector noise) is subtracted, leaving the peaks

above the noise at the base level.

Dalton (Da) According to the guidelines of the SI, the use of the

term Dalton for 1.6601  10

–27

kg is no longer

recommended. However it is still a current unit in

biochemistry.

Dendrogram A tree diagram to display data sets partitioned into

clusters.

Expression proteomics The massive parallel study of highly heterogeneous

protein mixtures with high throughput techniques

like 2-D electrophoresis and MALDI mass spec-

trometry or MDLC-ESI MS.

Functional proteomics This research is only possible with non-denatured

cell extracts and requires different tools than 2-D

electrophoresis and MALDI MS. A smaller subset

of proteins is isolated from the highly heteroge-

neous protein lysate and analyzed with mild tech-

niques that do not affect protein complexes and

three-dimensional structures.

Heat map A two-dimensional diagram to display data sets par-

titioned into clusters indicated by false color repre-

sentation.

Glossary of Terms

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

Glossary of Terms

Term Definition

False discovery rate

(FDR)

Statistical tool to determine new thresholds for

large numbers of Student’s t-tests, to be 95% sure

that a change is real.

K-means Statistical algorithm for clustering. In proteomics it

can be employed to produce clusters of proteins

which vary in similar ways over time.

Laboratory workflow

system

Database and computer network for the integrated

laboratory to control the entire workflow in the pro-

teomics factory.

Molecular mass (M

) The relative molecular mass is dimensionless. In

practice and in publication the dimension Da (Dal-

ton) is used. Particularly in electrophoresis the term

“molecular weight” is frequently used.

Normalization All peaks are reported with peak heights relative to

the highest peak height or area in the spectrum.

Optical Density (O.D.) The unit O.D. for the optical density is mostly used

in biology and biochemistry and is defined as fol-

lows: 1 O.D. is the amount of substance, which has

an absorption of 1 when dissolved and measured in

1 mL in a cuvette with a thickness of 1 cm.

Post-translational

modification (PTM)

Over 800 modifications of proteins have been iden-

tified, common examples include phosphorylation

and glycosylation. PTM analysis is an integral part

of proteomics.

Principal component

analysis (PCA)

Statistical tool for visualization of multidimensional

data by reducing the dimensionality of the data set.

Proteome The complete profile of proteins expressed in a giv-

en tissue, cell or biological system at a given time.

Proteomics Systematic analysis of the protein expression of

healthy and diseased, or treated and untreated cells,

tissues or biological fluids.

Quantification In many papers the term “quantitation” is used,

which is incorrect. In gel electrophoresis only rela-

tive quantification is possible, but the adjective “rel-

ative” is omitted in most papers on this subject.

Surface plasma

resonance

A technique which measures biomolecular binding

events in real time without the use of labels and has

become an established technique for measuring

biomolecular interactions

462

Glossary of Terms

Term Definition

Student’s t-test A statistical tool to check, whether the means of two

groups are statistically different from each other.

Electrophoresis

Analytical 2-D electro-

phoresis

Proteins are loaded in amounts of 10–100 lg.

Mostly broad pH intervals are used in the first

dimension.

Electroendosmosis

(EEO)

In an electric field, fixed charges on the gel matrix or

on a glass surface are attracted by the electrode of

opposite sign. As they are fixed, they cannot migrate.

This results in a compensation by the counterflow of

ions towards the cathode for negative or OH

ions towards the anode for positive charges. In gels,

this effect is observed as a water flow.

Immobilized pH

gradients

Polyacrylamide gels, which contain an in-built pH

gradient, created by acrylamide derivatives, which

carry acidic and basic buffering groups. Because an

immobilized pH gradient is absolutely continuous,

narrow pH intervals can be prepared, which allow

unlimited resolution.

Isoelectric point (pI) The pH value where the net charge of an ampho-

teric substance is zero. Because the pK values of

buffering groups are temperature-dependent, this is

valid also for the pI. The pI of a protein that can be

measured.

Phosphate-buffered

saline (PBS)

140 mol/L NaCl, 2.7 mmol/L KCl, 6.5 mmol/L

HPO

, 1.5 mmol/L KH

, pH 7.4.

Preparative 2-D electro-

phoresis

Proteins are loaded in the lower mg amounts.

Mostly narrow pH intervals are used in the first

dimension.

Rehydration The correct word is “rehydratation". Because this

word is a tongue-twister and is used many times for

the methodical description of the first dimension

separation, the incorrect term “rehydration” is gen-

erally used.

Two-dimensional

electrophoresis

There are more than one possibility to combine two

different electrophoretic separation principles. If

not further specified, 2-D electrophoresis means

isoelectric focusing under denaturing conditions

followed by SDS polyacrylamide gel electrophoresis.

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Glossary of Terms

Term Definition

Western blotting Electrophoretic transfer of separated proteins from

an electrophoresis gel onto the surface of a protein

binding membrane for immuno-detection.

Chromatography

IMAC IMAC is a technique for the enrichment of phos-

phopeptides from complex mixtures.

Mass Spectrometry

Adduct peak Results from the photochemical breakdown of the

matrix into more reactive species, which can add to

the polypeptide. Can also result from salt ions, Na

etc., that are embedded in the matrix.

Average molecular

weight

<2093.8>

avera

e molecular wei

The mass of a molecule of a given empirical for-

mula calculated using the average atomic weights

for each element. An average mass is obtained in

MALDI-TOF-MS when a peak is not isotopically

resolved (see mono-isotopic molecular weight).

AQUA A method for absolute quantitation, employing syn-

thetic peptides containing stable isotopes.

Base peak The most intense peak in a mass spectrum. A mass

spectrum is usually normalized so that this peak

has an intensity of 100%.

Bottom-up proteomics A strategy for protein ID within proteomics. In this

approach separated proteins or complex protein

mixtures are digested and the resultant peptides

analyzed by MS in order to identify the native pro-

tein.

Calibrant A compound used for the calibration of an instru-

ment.

Calibration A process where known masses are assigned to

selected peaks. The purpose is to improve the mass

accuracy of an MS instrument.

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Glossary of Terms

Term Definition

Centroided mass peak The centroided mass peak is located at the weighted

centre of mass of the profile peak.

Collision induced

dissociation (CID)

A process whereby an ion of interest, the precursor

ion, is selected, isolated, excited and fragmented by

collisions with an inert gas within the mass spec-

trometer.

Daughter ion (see

product or fragment ion)

An ion resulting from CID performed on a precur-

sor ion during a product ion MS/MS spectrum.

Digestion Cleavage of subject protein by proteolytic enzymes,

including trypsin and chymotrypsin.

Dried droplet method Sample preparation method for MALDI-TOF MS

applicable to peptides, protein digests, and full-

length proteins.

Electrospray ionization

(ESI)

An ionization technique which enables the forma-

tion of ions from molecules directly from samples

in solution. The ions formed in this process are pre-

dominantly multiply charged. Commonly coupled

with analyzers capable of tandem mass spectrome-

try (MS/MS). It is readily coupled with HPLC or

capillary electrophoresis.

Electron capture

dissociation

An alternative fragmentation method to CID, which

generates peptide and protein sequence informa-

tion, but is restricted to FT-ICR MS

Electron transfer

dissociation

A new fragmentation technique, non-ergodic in na-

ture, that fragments peptides by transferring elec-

trons to positively charged peptides and generates

peptide sequence information

External calibration A calibration is performed with a known calibration

mixture. The resultant calibration constants (file)

are then applied to a separate sample.

Fragment ion (product

or daughter ion)

See product ion.

Fragmentation A physical process of dissociation of molecules into

fragments in a mass spectrometer. The resultant

spectrum of fragments is unique to the molecule or

ion. Fragmentation data can be used to sequence

peptides and resultantly provide data for protein

identification.

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Glossary of Terms

Term Definition

Full length protein An intact polypeptide chain, constituting a protein

in its native or denatured state. The molecular

weight of which can be determined accurately with

MALDI and ESI MS.

ICAT Site specific labelling of cysteine residues with iso-

tope-coded affinity tags for quantitative analysis

In-gel digestion The embedded protein in the gel is cleaved using

enzymes of known specificity. During the process,

peptides are formed, which are extracted from the

gel for subsequent analysis.

Internal calibration Calibration where known masses in each spectrum

are used to calibrate that spectrum. Greater mass

accuracy than an external calibration.

Ion detector A detector that amplifies and converts ions into an

electrical signal.

Ion gate Typically an electrical deflector that permits certain

ions through to later stages of ion optics (open), or

deflects unwanted ions out of the way of the later

stages of the mass spectrometer. Commonly used

in post-source decay (PSD) analysis for the selection

of a precursor ion.

Ion source Region of the mass spectrometer where gas phase

ions are produced.

Ion transmission

efficiency

Refers to the fraction of the ions produced in the

source region that actually reaches the detector.

Ionization The process of converting a sample molecule into

an ion in a mass spectrometer.

Isotope Atoms of the same element having different mass

numbers due to differences in the number of neu-

trons.

Isotope abundance The relative amount in nature of certain atomic iso-

topes.

ITRAQ labeling of up to primary amino groups with isoba-

ric mass tags for quantitative analysis.

Linear time-of-flight

(TOF) mass analyzer

Simplest TOF analyzer, consisting of a flight tube

with an ion source at one end and a detector at the

other.

Mass accuracy The ability to assign the actual mass of an ion. This

is typically expressed as an error value.

466

Glossary of Terms

Term Definition

Mass analyzer Second part of the mass spectrometer, separating

the ions forms in the ion source according to their

m/z value. Examples of mass analyzers include ion

trap, quadrupole, time-of-flight and magnetic sec-

tor.

Mass range The area of interest to be measured in an experi-

ment. Or the capability of the analyzer.

Mass spectrometer An instrument that measures the mass to-charge

ratio (m/z) of ionized atoms or molecules. Com-

prises three parts: an ion source, a mass analyzer,

and an ion detector.

Mass spectrometry (MS) A technique for analyzing the molecular weight of

molecules based upon the motion of a charged par-

ticle in an electric or magnetic field.

Mass spectrum A plot of ion abundance (y-axis) against mass-to-

charge ratio (x-axis).

Mass-to-charge ratio

(m/z)

A quantity formed by dividing the mass of an ion

(Da) by the number of charges carried by the ion.

Matrix Necessary for the ionization of sample molecules by

MALDI. A small, organic compound which absorbs

light at the wavelength of the laser.

Matrix-assisted laser de-

sorption/ionization

(MALDI)

Ionization technique, commonly used for the ion-

ization of biological compounds. Sample is incorpo-

rated into the crystal structure of the matrix and

irradiated with the light from a laser.

Metastable ion An ion that decomposes into fragment ions and/or

neutral species, during its passage through the

mass spectrometer.

or MS/MS/MS Multistage tandem mass spectrometry where n is

greater than 2 and is used for further characteriza-

tion, such as identifying sites of phosphorylation.

467

Glossary of Terms

Term Definition

Monoisotopic molecular

weight

2092.6

monoisotopic molecular wei

The mass of a molecule containing only the most

abundant isotopes, calculated with exact atomic

weights. With respect to peptide analysis, the

mono-isotopic peak is the

C peak, i.e. the first

peak in the peptide isotopic envelope.

Multiple-charged ion Ion possessing more than a single charge. Charac-

teristic of ESI.

Neutral loss scan A type of MS/MS experiment. Useful for the indica-

tion of individual components in a complex mix-

ture.

N-terminal amino acid The amino acid residue at the end of a polypeptide

chain containing the free amino group.

Parent ion (precursor

ion)

Refers to the peak of an ion that will be selected for

fragmentation in a product ion MS/MS or PSD

spectrum.

Newton’s rings Interference pattern caused by light reflection be-

tween a flat surface surface and an adjacent imper-

fectly flat (slightly spherical) surface. It appears as a

series of concentric rings centered at the point of

contact.

Peptide-mass finger-

printing

Technique for searching protein databases for pro-

tein ID. Subject protein is cleaved and the resultant

cleaved peptide masses are used for a database

search.

Peak area The area bounded by the peak and the base line.

Can be calculated by integrating the abundances

from the peak start to the peak end.

Peak height The distance between the peak maximum and the

baseline.

Peak resolution The extent to which the peaks of two components

overlap or are separated. Compare with FWHM.

468

Glossary of Terms

Term Definition

Peak width The width of a peak at a given height.

Post-source decay (PSD) A technique describing fragmentation of a precur-

sor ion that occurs in the first field free region of

the TOF before the reflectron.

Precursor ion (parent

ion)

Ion selected to undergo fragmentation within the

mass spectrometer in a product ion MS/MS or PSD

spectrum.

Precursor ion scan A type of MS/MS experiment. Useful for the indica-

tion of individual components in a complex mix-

ture.

Product ion (see

daughter ion)

An ion resulting from CID performed on a precur-

sor ion during a product ion MS/MS spectrum.

Product ion scan The principle MS/MS experiment. Involves the

selection of a precursor ion to undergo fragmenta-

tion within the MS.

Protein characterization The identification of structural aspects of a protein.

Includes amino acid sequence, molecular weight,

three-dimensional structure, post-translational

modifications and biologic activity of a particular

protein.

Protein sequencing The determination of the order of amino acids in a

subject protein or peptide.

Pulsed mass analyzer Includes time-of-flight, ion cyclotron resonance,

and quadrupole ion traps. An entire mass spectrum

is collected from a single pulse of ions.

Reflectron Improves resolution of a mass spectrometer by act-

ing act as an ion mirror. Compensates for the distri-

bution of kinetic energy, ions of the same mass

experience in the source.

469

Glossary of Terms

Term Definition

Resolution

10 % valley

10 %

m∆

Refers to the separation of two ions where resolu-

tion, R = m/m. For a single peak made up of singly

charged ions at mass m in a mass spectrum, the

resolution maybe expressed as m/where m is the

width of the peak at a height that is a specified frac-

tion of the maximum peak height (for instance full

width at half maximum height; FWHM). A second

definition for defining resolution is 10% valley. Two

peaks of equal height in a mass spectrum at masses

m and m–m are separated by a valley that at its low-

est point is just 10% of the height of either peak.

Resolving power (mass)

5000

5001

mass resolution

The ability to distinguish between ions differing

slightly in mass-to-charge ratio.

Sample preparation A crucial stage to achieve efficient, optimal ioniza-

tion.

SDS electrophoresis Proteins are separated in a polyacrylamide gel as

negatively charged protein–detergent micelles. Sec-

ondary, tertiary, quaternary structures and the indi-

vidual charges of the proteins are cancelled. The

migration distances of the resulting zones from the

origin correlate roughly to the logarithm of the M

of the respective protein.

Seeded microcrystalline

film method

A sample preparation method. First, a thin layer of

small matrix crystals is formed on the sample slide.

Then a droplet containing the analyte is placed on

top of this layer. This is left to dry. The deposit is

washed before the sample slide is inserted into the

mass spectrometer. Is a direct replacement of the

dried droplet method.

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