Mass spec lecture

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)

How does it work?

• Gas-phase ions are separated according to mass/charge ratio and sequentially detected

Parts of a Mass Spec

• Sample introduction

• Source (ion formation)

• ________________________

• Mass analyzer (ion sep.) high vac

• Detector (electron multiplier tube)

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)

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

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

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

]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abu

ndan

ce

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

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

Mass Analyzers• Low resolution

– Quadrupole– Ion trap

• High resolution– TOF time of flight– Sector instruments (magnet)

• Ultra high resolution– ICR ion cyclotron resonance

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

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

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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

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

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?

Quadrupole Mass Ion Filter

Ion Trap

Time of Flight -TOF

                                                      

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)

TOF with reflectronhttp://www.rmjordan.com/tt1.html

Sector instrumentshttp://www.chem.harvard.edu/mass/tutorials/magnetmovie.html

FT-ICRMS

• http://www.colorado.edu/chemistry/chem5181/MS_FT-ICR_Huffman_Abraham.pdf

CID or MS-MS

– MS-MS • sequencing the peptides or

oligonucleotides• structural characterization of drugs and

metabolites • Assay development, sensitivity

enhancement

HPLC-MS-MS

time

response

chromatogram

m/zm/z

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

HPLC-MS

• Reverse-phase HPLC

–Separation of involatiles (peptides)

–The lower the flow the greater the sensitivity

–Column ID (300 m – 50 m)

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)

0 5 10 15 20 25 30 35 40 45

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

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

400 600 800 1000 1200 1400 1600 1800

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

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)+

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

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

L

SY

EY

LF

S

G

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

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

LC-MS-MS analysis

• Characterization of synthetic processes

• Drug metabolism studies – structural elucidation of metabolites

• Quantification of polar molecules in biological samples – NAD

NAD Assay

• Goal to determine the relative importance of the different biosynthetic pathways.

• Stable isotopic incorporation

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

+ + +

*

* *

*

*

*

* *

**

*

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

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

0

1

2

3

4

5

6

0 100 200 300 400 500 600 700 800

Concentration of NAD (ng/ul)

Standard Curve

• 39 pts• Detection Limit: 5 pg/l or 35 fmol on column

8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.50

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

660 665 670 675 680 685 690 6950

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

664

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

Elemental Mass Spectrometry

• ICP-MS (inductively coupled plasma)

• SIMS-TOF (secondary ion mass spectrometry

• CRIMS

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

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