ABSTRACT
Isotopes: • Species of elements with differing numbers of neutrons, e.g., 16O, 17O, 18O;
most isotopes are stable but some are radioactive, e.g., 14C • Some halogens, Cl and Br, and metals, e.g., Sn, Pt, and Pd, significantly
alter the isotope patterns of small molecules
• Contributions of carbon isotopes may be significant for large biological compounds; there is a 1.1% chance that any given carbon in a compound will be a 13C. Once there are more than 91 C atoms (proteins contain thousands of C), the peak(s) containing 13C will be more intense than those containing only 12C
m, mass:
• Unit is dalton (Da); 1 Da = 1/12 of the mass of a 12C atom
z, unit of charge:
• Unit of charge on a proton (positive) or an electron (negative), expressed (arbitrarily) in whole numbers, i.e., +1 or –1
m/z, mass-to-charge ratio:
• A measured property of an ion • m/z is dimensionless; usually (but not necessarily) z = 1 • In electrospray, z increases with molecular mass
Nominal mass:
• Calculated, based on isotopic masses of the elements using integer masses, e.g., H = 1, O = 16
Exact mass:
• Calculated, based on isotopic masses using unified mass units based on C = 12.0000; e.g., H = 1.007825, O = 15.9949
Accurate mass:
• Measured mass of an analyte; value determined to the third or fourth decimal place
Monoisotopic mass:
• Calculated, based on the masses of the most frequently occurring isotopes
Molecular mass:
• Calculated, based on average atomic masses of the elements (all isotopes averaged), e.g., C = 12.0108 Da, O = 15.9994 Da
Isobaric mass:
• Empirical formulae that have the same nominal mass but different exact masses
MS or ms may mean (depending on context):
• Mass spectrometry, mass spectrometer, mass spectrometric, or mass spectrum (singular or plural)
Mass spectrometer:
• An analytical instrument that produces a beam of gas phase ions from samples, sorts the resulting mixture of ions according to their mass-tocharge (m/z) ratios using electrical or magnetic fields (or combinations thereof), and provides analog or digital output signals (peaks) from which the m/z and the intensity (abundance) of each detected ionic species may be determined
Mass spectrometers operate on the premises that:
• The sample is in the gas phase and is ionized (neutral molecules cannot be manipulated with electrical or magnetic fields)
• The ions are separated according to their m/z and then detected
Mass spectrometers obtain information on:
• What is present and how much is present by determining ionic masses and their intensities
Mass spectrometers measure:
• m/z not m
Components of a mass spectrum:
• A spectrum is a two-dimensional plot with m/z (not m) on the x-axis and intensity (usually relative) on the y-axis
• Depending on the ionization method, the molecular ion may be [M]+, e.g., in EI, but adducts are formed in soft ionization, e.g., [M + H]+, [M + adduct]+ in ESI
• Other ions in a spectrum are fragments • The most intense ion is the base peak • Less intense ions (usually to the right of the major ions) are isotope peaks
(mostly attributable to 13C)
Diversity of MS:
• All spectroscopies, e.g., UV and IR, examine an invariate parameter of a molecule
• In mass spectrometry the ability to control where and how much energy is added to a molecule is widely variable and selectable, depending on the application, i.e., for structural characterization or quantification
• Added energy is determined/controlled in the ion source and in CID cells
• Qualitative for confirmation/identification, quantitative for selected constituents
Functions: • Produce vapors from samples; introduce a sufficient quantity into the ion
source so that its composition represents that of the original sample
Polarity is important for analytes of ≤1 kDa: • Affects the selection of the sample introduction method • Depends on the number of primary amino, hydroxyl, and acidic groups
Gas chromatography (GC): • Samples must be nonpolar and remain stable when volatilized in helium
(up to 320°C) • Good to ∼600 Da
Liquid chromatography (LC): • Polar samples acceptable; atmospheric pressure ionization (API) methods
enable interfacing with reversed phase columns • Components are separated as the mobile phase is changed from aqueous to
organic, with polar compounds eluting first • Good to 100 kDa
Capillary electrophoresis (CE): • Specific form of LC • Efficient for large biomolecules • Low flow rates make interfacing difficult
Semipermeable polymer membrane: • Different permeabilities enable passage of volatile organic compounds
through a silicone membrane (relative to water or inorganic gases) • Selective introduction of certain analytes present in aqueous or gaseous
streams
Other sample introduction methods: • Batch inlet • Direct insertion probe • Atmospheric solids analysis probe
Functions: • Produce an ion beam representative of the sample; then form, shape, and
eject the beam into the mass analyzer
Hard ionization, such as electron ionization (EI): • Imparts more energy than needed to form molecular ions • Extensive fragmentation occurs because the excess energy causes dissocia-
tion of bonds; molecular ion may be lost
Electron ionization (EI): • Takes place inside vacuum chamber • Energetic (70 eV) electron beam from a heated filament impacts vaporized
analytes, displaces an electron, and generates radical cations that frequently fragment
• Reproducibility of spectra is excellent; detection limits are low • Mass limit 1 kDa • Mostly used in GC-MS
Soft ionization (CI, API, MALDI): • Added energy is sufficient only to generate adducted ions, e.g., [M + H]+
• Ions formed are protonated molecules (not protonated molecular ions)
Energy transfer: • Highest to lowest: EI > CI ≈ APCI ≈ APPI > MALDI ≈ ESI
Currently most important: • ESI and MALDI, especially for biological (polar) analytes
Chemical ionization (CI): • Takes place inside vacuum chamber • Electrons from heated filament react with a reagent gas (e.g., methane)
forming ions that in turn transfer a proton to the analytes, producing protonated molecules, [M+H]+
• Particularly useful for quantification • Mass limit 1 kDa • Mostly used in GC-MS
Atmospheric pressure ionization (API): • Includes ESI, APCI, and APPI • Ionization takes place outside vacuum chamber • Enables simultaneous vaporization and ionization of polar compounds • Predominant ions are [M + H]+ and [M + adduct]+
Electrospray ionization (ESI): • Analytes in solution are sprayed from a stainless tube held at 2-5 kV, drop-
lets are dried, ions are released through charge repulsion • Favors most polar analyte in a mixture • Molecules of >1 kDa form multiply charged ions appearing as an envelope
(skewed to lower m/z) that must be deconvoluted (transformed) to obtain the molecular mass of the neutral, zero charge state
Atmospheric pressure chemical ionization (APCI): • Equivalent of CI at atmospheric pressure • Corona discharge is used for ionization (instead of electrons from a filament) • More uniform than ESI, but analytes must be polar • Mass limit 1 kDa
Atmospheric pressure photo-ionization (APPI): • Similar to APCI • UV source used instead of corona discharge • Extends ionization to compounds that are less polar than can be ionized
with APCI, e.g., aromatic hydrocarbons
Surface ionization methods: • Take place outside vacuum chamber at ambient pressure and temperature • Include DESI (and related methods) and DART • Mainly qualitative analysis of compounds adsorbed on surfaces
Desorption electrospray ionization (DESI): • Spray of charged droplets (methanol, water) bombards a surface, ejecting
and ionizing analytes • No sample preparation, analysis in situ • Cannot be combined with chromatography
Direct analysis in real time (DART): • Ionization using an excited gas stream (He) that bombards the sample;
simple, rapid • Cannot be combined with chromatography
Matrix-assisted laser desorption/ionization (MALDI): • Sample is mixed with ∼1,000-fold excess of a special matrix (aromatic acid) • Spotted and dried on a metal plate, placed in vacuum chamber • Matrix absorbs the energy when irradiated with a UV laser; analyte and
matrix evaporate • Ions (singly charged) are formed at the surface or during evaporation • Almost always analyzed using TOF
Function: • Separate ions according to their m/z
Classification of analyzers: • Based on whether or not the operating parameters must be altered to observe
ions with differing m/z values • In scanning instruments (quadrupoles, traps, magnetic) electric or magnetic
fields must be varied continuously to obtain spectra • In nonscanning analyzers (TOF, FT-based) spectra are collected without
changing operating parameters
Quadrupole (Q): • Molybdenum rods (10-20 cm long, 1 cm diameter) placed in a square with
opposite pairs connected electrically • Mass separation (filtering) takes place when ions oscillate in the field pro-
duced by superimposed rf and dc voltages • For any given field only one specific m/z has a stable trajectory and can
move to the detector; all other ions describe unstable paths and discharge onto the rods
• Mass range is scanned by changing rf and dc voltages progressively while keeping their ratio constant
• Selected ion monitoring (SIM) uses a single rf/dc field • Qs have unit resolution, mass range is to 4 kDa • Compatible with GC-MS and LC-MS • Quadrupole (q): in rf only mode acts as ion transmission device
Time-of-flight analyzer (TOF): • Pulses of ions are accelerated from the source into an analyzer tube where the
time is measured for an ion to travel through a field free region to the detector • The time-of-flight is a function of the momenta of ions, and therefore of
their m/z; the acceleration voltage, and thus the kinetic energy (momentum), is the same for all ions, so those with the lowest m/z will travel fastest and arrive at the detector first, followed by the sequential arrival of ions with successively higher m/z
• Delayed extraction reduces spatial distribution by aligning and concentrating ions at an electronic gate prior to their release into the analyzer (MALDI)
• Orthogonal injection is used to create ion pulses in ESI • Reflectrons improve resolution and mass measurement accuracy by account-
ing for differences in the energy of ions that have the same empirical formula • Resolution to 60,000 • Upper mass limit 350 kDa, theoretically unlimited • Compatible with GC-MS and LC-MS
Orbitrap: • Electrostatic ion trap comprised of a spindle-shaped inner electrode and a
split outer electrode; ions (from API) rotate around and oscillate along the inner electrode
• Oscillating ions come close to the split outer electrode, inducing a current, called a transient, that is interpreted using Fourier transform (FT) analysis
• No superconducting magnet • Resolution up to 250,000, accurate mass measurement, dynamic range >103
• Compatible with LC-MS but not at very high resolution
Fourier transform ion cyclotron resonance (FT-ICR): • Based on the fact that ions move in circular paths in a magnetic field • Frequency of rotation depends on the m/z of ions and strength of the mag-
netic field; direction of ion motion is such that a positive ion traveling through a magnetic field is set at 90° to the trajectory curves in a clockwise direction; radius of the curve is proportional to ionic mass (left-hand rule)
• Resolution up to 3 million, very accurate mass measurement (<1 mDa) • Not compatible with LC-MS at highest resolution
Ion mobility separator (IMS): • While MS characterizes ions according to m/z, IMS adds another dimen-
sion by enabling investigation of the shape of ions • Ions enter a cell carried by voltage waves but counteracted by a flow of nitrogen • Of two isomers, the one with more open form (greater cross-sectional area)
will be slowed by the counterflow and emerge later than its more compact counterpart
Magnetic sector: • Ions are accelerated by a fixed voltage; a specified angle of movement is
required for ions to reach the detector • Scanning the strength of the magnetic field systematically allows each ion with
a given m/z to achieve the necessary angle of deflection and reach the detector • Scan times are slow • Used only for specific applications
Multianalyzer system (MS/MS): • Now common to combine two or more analyzers within a single instrument
(MS/MS) to improve and extend analytical capabilities • Tandem-in-space combinations involve similar analyzers, tandem instru-
ments, or use mixed types, hybrid instruments • Common tandem MS/MS: triple quadrupole (QqQ) and TOF-TOF • Common hybrid MS/MS: QTOF, LIT-orbitrap, and LIT-FTICR • Tandem-in-time analyzers use the same analyzer twice (QIT and LIT)
• MS/MS investigates both mass and structure of ions: first analyzer is used to select an ion of interest that is passed, usually after fragmentation, into the second analyzer, often of higher resolving power, providing accurate mass measurement
Triple quadruple (QqQ): most versatile of MS/MS instruments: • Used for product ion, precursor ion, and neutral loss scans as well as for
selected reaction monitoring
Quadrupole ion trap (QIT): • Three-dimensional Q; all ions maintained in stable trajectory (complex
sinusoidal path) within the trap • Selected m/z values are detected as they are rendered unstable by an applied
voltage and ejected onto the detector • Specified ions maintained in the trap with stable trajectories for sequential
MS/MS analyses (MSn) • Limited number of ions because of space-charge effects
Linear ion trap (LIT): • Similar to QIT, but ions oscillate in linear fashion along the length of
a quadrupole • Linear format removes most crossover points where major space-charge
problems are encountered in QIT, thus improving sensitivity because more ions stored
Quadrupole time-of flight (QTOF): • Quadrupole, used in Q or q mode, transmits ions to CID cell; resulting frag-
ments are analyzed by TOF • Accurate mass data
Linear ion trap-orbitrap: • LIT can be used for MS or MS/MS • Ions from LIT pass to orbitrap for accurate mass measurement • May have second collision cell for high-energy CID
Linear ion trap-FTICR: • LIT can be used for MS or MS/MS • Ions passed to ICR cell for accurate mass measurement or IRMPD/ECD
(fragmentation methods in ICR cells)
Function: • Detection of ions emerging from the analyzer
Arriving ions form very small current: • One ion per second is 1.6 × 10-19 A • Ion currents usually 10-9 to 10-16 A • In FT instruments ions induce a transient signal
Electron multiplier: • Arriving ions instigate an electron cascade increasing the signal about
a million-fold • Resulting signal is amplified electronically and recorded by the data system
Types of multipliers: • Continuous dynode, discrete dynode, multichannel plate (MCP); photomul-
tiplier detector (Daly)
Orbitrap and ICR: • Ion detection is based on generated image currents • The passage of an ion close to a metal surface induces an alternating elec-
tric current (transient) • Frequency is proportional to m/z of the ion • FT analysis is used to deconvolute the transient and obtain spectra
Functions: • Prevent loss of ions by collision with neutral molecules (air) in evacu-
ated chambers • Remove unreacted molecules from ion source to prevent memory effects,
e.g., from chromatographic effluents
Mean free path: • Average distance that an ion can travel before encountering a neutral molecule • May be kilometers/miles in ICR, necessitating vacuum of 10-10 Torr
First stage of pumping (rough vacuum): • Rotary or scroll pumps • Initially removes atmospheric gases (to 10-2 Torr) • Continuously removes exhaust gases from high-vacuum pumps
Second stage of pumping (high vacuum): • Turbomolecular pumps (spinning at 50,000-90,000 rpm); 10-5 to 10-10 Torr
Differential pumping: • Maintains necessary vacuum in different chambers of the instrument,
e.g., between CI source and analyzer
Functions: • Control operational processes • Acquire and process generated data (e.g., thresholding, centroiding) • Interpret data locally • Post-process data (databases, Internet)
Resolution: • Ability to differentiate one mass from another • Defined with respect to the mass and width of a peak at half its maximum
height; e.g., if peak for an ion at m/z 800 is 0.05 wide resolution is 16,000
Resolving power: • The inverse of resolution
Analyzers have different resolutions: • Quadrupoles have unit resolution throughout mass range, e.g., m/z 200 from
201 and 2000 from 2001 • TOF, orbitrap, and ICRMS up to 60,000, 250,000, and 3,000,000, respectively
Resolution in TOF: • Function of design • Data collection time makes no difference • Harder to achieve high resolution at low masses because peaks are narrow
Resolution in FT: • Time dependent • For example, in an ICR, to obtain a resolution of 500,000 at m/z 800 with a
9.8 Tesla magnet, a 5 s transient is required • Not compatible with chromatography when peaks are a few seconds wide • More difficult to reach high resolutions as the mass of the analytes increases
Accurate mass measurements: • Easier at high resolution because isobaric (same nominal mass) species
are resolved • Resolution does not ensure accuracy, because the latter depends on the cali-
bration of the mass scale
• FT-ICRMS provides highest accuracies using a combination of accurate calibration, continuous data collection, and very high resolution
• Achievable with analyzers having moderate resolutions, e.g., 5,000, if there are no interferences from adjacent peaks
Accurate mass (applications and limits): • Obtain empirical formula to assist in identification of small molecules • Assignment of amino acid sequences and deconvolution of overlapping
chromatographic peaks in tryptic digests of proteins • Number of possible empirical formulae increases dramatically with m/z • Number of possible empirical formulae increases for a specified m/z when
the number of elements is increased, e.g., adding S, Si, Cl, and Br to the C, H, O, and N
Difference between calculated (exact) and experimentally determined (accurate) masses:
• Expressed in millidaltons (mDa), millimass units (mmu), or in parts per million (ppm)
• Highest observable for a given instrument • Not directly related to resolution • Poor resolution at high mass is undesirable
LOD: • Minimum amount of an analyte that can be observed reliably • Often defined as 3× background noise
Instrument LOD: • Minimum amount of analyte observable
Sample LOD: • Minimum amount of analyte observable after extraction from a matrix
LOQ: • Minimum amount of an analyte that can be quantified reliably • Often defined as 3× LOD
• Spectra for confirmation of identity or identification of unknowns • Quantification (often with selected ion monitoring)
• Used in MS/MS
CID (fragment) ions:
• Generated in a collision cell between two analyzers; selected precursor ions from MS1 are accelerated into a collision (target) gas
• Multiple collisions additively increase the internal energy of the ions and cause fragmentation
• Product ions analyzed in MS2
Triple quadrupoles:
• Most important use of CID is in selected reaction monitoring (SRM) for quantification
Peptides:
• CID fragmentation of bonds yields b and y ions that enable sequencing • Drawback: phosphate post-translation modifications (PTMs) are lost • Alternative fragmentation methods, electron transfer dissociation (ETD),
and electron capture dissociation (ECD) preserve phosphates • Drawback: large multiply charged (z > 2) peptides required
Infrared multiphoton dissociation (IRMPD):
• Equivalent of CID in FT-ICRMS
Selected ion monitoring (SIM):
• Single quadrupoles • Analyzer voltages set to monitor selected single m/z
Selected reaction monitoring (SRM):
• Triple quadrupoles • Analyte m/z is selected in Q1 subjected to CID in q2 and a chosen m/z is
monitored with Q3
Specificity and sensitivity:
• Better in SRM than in SIM
Calibration curves:
• Regression analysis used to fit equation to standards and determine unknown concentrations of analytes with known identities, e.g., pharmaceuticals
External standard method: • Series of individual (blank) samples spiked with increasingly higher con-
centrations of analyte
Internal standard (IS) method: • Similar to external method but adding the same amount of a structurally
similar IS to each sample prior to extraction • Compensates for recovery differences • Stable isotope labeled analytes are the best IS
Standard addition method: • Requires multiple sample aliquots of the sample • First aliquot used as is • Subsequent aliquots are supplemented with increasing (known) amounts of
the analyte
Small molecule analysis: • Phthalates (plasticizers), siloxanes (GC-MS), PEG
Biopolymer analysis: • Trypsin peptides, keratin peptides (human or animal); siloxanes and PEG
(from buffers)
• Frozen (10-20 μ) tissue sections analyzed by MALDI/TOF • Location of analytes within tissue, currently reaching cellular level • Many types of compound, most commonly lipids
Bottom-up: • After enzymatic digestion (trypsin), peptides are analyzed by LC-MS/MS • Combination of partial sequences and peptide masses for two or three pep-
tides sufficient for identification using databases (accessed using the Internet) • Hundreds of proteins may be identified in single analysis
Top-down: • Intact individual proteins analyzed by FT-ICRMS • Amino acid residues are removed in sequence; data are complex, as all
fragment ions form multiply charged envelopes • Must account for 13C • Post-translational modifications are preserved
De novo: • Used when there is no information available in databases • Entire amino acid sequence must be derived from mass spectra • Steps: digest multiple samples with several enzymes to produce differ-
ent sets of peptides; determine m/z ratios; use MS/MS to obtain amino acid sequences of each peptide; cross reference data sets to obtain overall sequence of protein.
