mass spec lecture. what kind of info can mass spec give you? molecular weight elemental composition...
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![Page 1: Mass spec lecture. What kind of info can mass spec give you? Molecular weight Elemental composition (low MW with high resolution instrument) Structural](https://reader036.vdocuments.net/reader036/viewer/2022081519/56649d575503460f94a365d0/html5/thumbnails/1.jpg)
Mass spec lecture
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What kind of info can mass spec give you?
• Molecular weight
• Elemental composition (low MW with high resolution instrument)
• Structural info (hard ionization or CID)
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How does it work?
• Gas-phase ions are separated according to mass/charge ratio and sequentially detected
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Parts of a Mass Spec
• Sample introduction
• Source (ion formation)
• ________________________
• Mass analyzer (ion sep.) high vac
• Detector (electron multiplier tube)
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Sample Introduction/Sources
Volatiles• Probe/electron impact (EI),Chemical ionization
(CI)• GC/EI,CIInvolatiles• Direct infusion/electrospray (ESI)• HPLC/ESI• Matrix Assisted Laser Adsorption (MALDI)Elemental mass spec• Inductively coupled plasma (ICP)
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EI, CI• EI (hard ionization)
– Gas-phase molecules enter source through heated probe or GC column
– 70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions
– EI spectra can be matched to library stdsCI (soft ionization)– Higher pressure of methane leaked into the
source (mtorr)– Reagent ions transfer proton to analyte
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CI/ ion-molecule reaction
• 2CH4 + e- CH5+ and C2H5
+
• CH5+ + M MH+ + CH4
• The excess energy in MH+ is the difference in proton affinities between methane and M, usually not enough to give extensive fragmentation
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Electrospray
• 5 kV voltage on a needle
• Nebulization gas
• Produces gas-phase protonated analytes
• Little to no fragmentation
• Multiple charging
• 10 M angiotensin at 5 l/min direct infusion, MW 1269
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]
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433.3649.2
1296.6
671.1
682.1647.5 1340.6
784.4534.4371.1 1362.51028.4214.0 1127.3 1440.1810.8 1743.0928.6 1525.6 1820.4 1989.01607.11160.0
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MALDI
• Matrix -UV absorber, ex. picolinic acid, cinnimic acid
• Singlely charged ions
• Need mass analyzer with a large m/z range – TOF
• Laser pulse as opposed to continuous source
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Mass Analyzers• Low resolution
– Quadrupole– Ion trap
• High resolution– TOF time of flight– Sector instruments (magnet)
• Ultra high resolution– ICR ion cyclotron resonance
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Resolution
• R = m/z/m/z
• Unit resolution for quad and trap
• TOF up to 15000
• FT-ICR over 30000– MALDI, Resolve 13C isotope for a protein that
weighs 30000– Resolve charge states 29 and 30 for a protein
that weighs 30000
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High vs low Res ESI
• Q-TOF, ICR– complete separation of the isotope peaks of a
+3 charge state peptide– Ion abundances are predictable– Interferences can be recognized and
sometimes eliminated
• Ion trap, Quad– Unit resolution
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MVVTLIHPIAMDDGLR594.3594.7
595.0
601.3
595.3601.0
601.7
602.0
m/z
C78H135N21O22S2+3
M/zth = 7.0M/z = 7.0
Q-TOF
901.4
891.7
902.3
900.6
891.2
892.6
MVVTLIHPIAMDDGLR
C78H135N21O22S2+2
Xcorr = 3.09
Mth = 10.5
M = 9.7
LCQ
R = 0.88
m/z
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Exact Mass Determination
• Need Mass Spectrometer with a high mass accuracy – 5 ppm (sector or TOF)
• C9H15NO4, FM 201.1001 (mono-isotopic)
• Mass accuracy = {(Mass Error)/FM}*106
• Mass Error = (5 ppm)(201.1001)/106 =
0.0010 amu
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Mass accuracy
• Mass Error = (5 ppm)(201.1001)/106 =
0.0010 amu
• 201.0991 to 201.1011 (only 1 possibility)
• Sector instruments, TOF mass analyzers
• How many possibilities with MA = 50 ppm?
with 100 ppm?
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Quadrupole Mass Ion Filter
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Ion Trap
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Time of Flight -TOF
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Where:
•mi = mass of analyte ion
•zi = charge on analyte ion •E = extraction field •ti = time-of-flight of ion
•ls = length of the source
•ld = length of the field-free drift region •e = electronic charge (1.6022x10-19 C)
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TOF with reflectronhttp://www.rmjordan.com/tt1.html
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Sector instrumentshttp://www.chem.harvard.edu/mass/tutorials/magnetmovie.html
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FT-ICRMS
• http://www.colorado.edu/chemistry/chem5181/MS_FT-ICR_Huffman_Abraham.pdf
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CID or MS-MS
– MS-MS • sequencing the peptides or
oligonucleotides• structural characterization of drugs and
metabolites • Assay development, sensitivity
enhancement
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HPLC-MS-MS
time
response
chromatogram
m/zm/z
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hybrids
• Ex. Q-TOF– Trap has excellent sensitivity (can store
essentially all ions), mass selectivity (can store ions of a particular m/z ratio
– TOF is a high resolution mass analyzer
• Triple quadruple– Neutral loss scan
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HPLC-MS
• Reverse-phase HPLC
–Separation of involatiles (peptides)
–The lower the flow the greater the sensitivity
–Column ID (300 m – 50 m)
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Proteomics using LC-MS• Protein identification, characterization,
quantification• Extract proteins, fractionate proteins
(typically using 2D-gel electrophoresis)• Digest protein(s) with a protease to
produce peptide mixture (lysine, arginine)• LC-MS-MS analysis• Database searching identifies proteins
– Mascot, Expasy (tools)
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11.36 17.23
12.57
12.74
17.68
36.21
1.21 15.13 24.95
24.53
22.462.54
3.01 21.735.43 6.14 25.20
20.41 48.5527.31 37.1829.53 32.43 40.11 45.43
RP-HPLC Separation of a Tryptic Digest of BSA
Peptide used in following illustration
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784.6
785.4
1568.6795.6
1567.5
1121.4
812.5
1122.5830.41280.41234.41064.4 1570.6852.5691.5 1365.5997.4 1180.3391.0 428.9 1477.0591.3 1706.0 1768.7
Mass Spectrum of a Tryptic Peptide from BSA
(M+2H)+2
(M+H)+
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D A F L G S F L Y E Y S R
N term C term
H2N
HN
NH
HN
NH
HN
O
GO
O
O
O L
F
A
D
S
O+
H2N
HN
NH
HN
NH
HN
NH
HN
D
O A
O F
O L
O G
O S
F
O L
O O
b ion (m/z 590)
b ion
y ion
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300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
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D A F L G S F L Y E Y S R
MS-MS Spectrum
D A F L G S+
m/z 590
Residue massS 87.0Y 163.1E 128.1L 113.1F 147.1G 57.0
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Peptide Mass Mapping
2211.37
1639.40
1750.43
1567.23
1480.26
1439.32
1305.29
1249.17
1193.16
1142.29
1083.21
1001.22
927.16
842.19
655.84
567.95MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGEEQFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVADESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEPERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGKYLYEIARRHP YFYAPELLYY ANKYNGVFQD CCQAEDKGACLLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVARLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDRADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDAIPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRRHPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKLKHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQVSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLILNRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETYVPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKPKATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVVSTQTALA
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LC-MS-MS analysis
• Characterization of synthetic processes
• Drug metabolism studies – structural elucidation of metabolites
• Quantification of polar molecules in biological samples – NAD
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NAD Assay
• Goal to determine the relative importance of the different biosynthetic pathways.
• Stable isotopic incorporation
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NAD Synthesized from Labeled Precursors
NAD synthesized from
a) 2H4 labeled nicotinic acid and nicotinamide (m/z 667)
b) 13C5 labeled tryptophan (m/z 669)
c) 13C6 labeled quinolinic acid (m/z 670)
N
CONH2D
D D
N
CONH2
N
CONH2
+ + +
*
* *
*
*
*
* *
**
*
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The Experimental Strategy
• Culture cells in media containing isotopically labeled precursors for fixed time intervals
• Harvest and lyse the cells, extract the NAD, and quantitate the unlabeled and labeled NAD using reverse-phase LC-MS-MS
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Sensitivity advantage
N
N
H2N
N
N
O
HO OH
O
P
O
P
OO O
O O
O
HO OH
N
CONH2-
+
N
N
N
N
O
HO OH
O
P
O
P
OO O
O O
O
HO OH
N
NH2
CONH2-
+
m/z 664 m/z 542
m/z 692 m/z 562
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Concentration of NAD (ng/ul)
Standard Curve
• 39 pts• Detection Limit: 5 pg/l or 35 fmol on column
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8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.50
20
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667
692
Unlabeled NAD
Internal Standard
NAD synthesized from labeled nicotinamide
Sample from experiment 1:Nicotinamide at 72 hrs
m/z 664
m/z 692
m/z 667
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Elemental Mass Spectrometry
• ICP-MS (inductively coupled plasma)
• SIMS-TOF (secondary ion mass spectrometry
• CRIMS
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ICP-MS (vs. ICP-UV/vis)• ICP (ch 10, pg 231-232) ICP-MS (ch 11)
• A spark ignites flowing argon forming a self-sustaining plasma (T ≈ 10000 K)
• Sample is aspirated/pumped into plasma forming elemental cations and some simple polyatomic ions
• Ions are pushed into mass analyzer by high voltage
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Isotope ratio mass spectrometry• Elemental analysis (geologists,
archeologist, isotope tracer studies)
• High resolution sector mass analyzers
• Faraday cups
• ThermoFinnigan Neptune http://www.thermo-optek.it/GetBrochure.php?ID=43
• CRI-MS (chemical reaction interface) – Converts all carbon to CO2
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