Mass Spectrometry • Widely used analytical technique• Within an accuracy of 0.01% of total weight of sample and within 5 ppm

for small organic molecules• Unequaled sensitivity –Nanomolar range routinely (1 x 10-9M) –Femtomolar range possible (1 x 10-15M) –Attomolar range claimed (1 x 10-18M)• Diversity of applications –Proteins –Oligonucleotides –Oligosaccharides –Lipids –Others• Proteomics –Identification of proteins expressed under specific conditions

-3 fundamental parts: Ionization source, the ana-lyzer, the detector

-Ionization source

시료분자를 이온화시키고 더 작은 이온으로 쪼갠다 .

-Mass analyzer

이온들을 mass-to-charge(m/z) ratio에 따라 선택적으로 분리

-Ion detector

이온 흐름을 그 양에 비례하게 전기적인 흐름으로 전환 , 증폭시켜 signal을 생성

-Vacuum system

Basic components to MS•Ion source –Electrospray(ESI) –Atmospheric Pressure Ionization (APCI) –Chemical Ionization (CI) –Electronic Ionization (EI) –Matrix Assisted Laser DesorptionIonization (MALDI)

•Mass Selection –Quadrupole –Time of Flight (TOF) –Magnetic Sector –Ion Trap –Ion Cyclotron

•Detector –Phosphor / Photo Diode –Multi-channel Plate (MCP)

Ion Source: ESI

Electrospray ionization(ESI)- 용액 상태의 시료를 이온화 (LC-MS)- 기존의 방법으로는 얻기 힘들었던 intact

상태의 peptide 나 단백질을 이온화- 한 개 이상의 전하를 띤 이온을 생성

Ion Source: ESI

Ion Source: ESI

Ion Source: MALDI

Matrix Assisted Laser Desorption Ion-ization(MALDI)

Laser

matrix + analyte

Sample support

a

m

m

m

m

m

m

mm a

a

a

a

+

+

+

+m

am+

Matrix

HO

CH C(CN)COOHOH

HO

COOH

HO

CH3O

CH3O

CH CHCOOH

-cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid(2,5-DHB)

Sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid)

HO

CH C(CN)COOHOH

HO

COOH

HO

CH3O

CH3O

CH CHCOOH

-cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid(2,5-DHB)

Sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid)

hn

Laser

+20 kV

Variable Ground Grid Grid

AH+

Sample plate 1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI plate.

2. Laser flash ionizes matrix molecules.

3. Sample molecules are ionized by proton transfer from matrix:

MH+ + A M + AH+.

Why MALDI?

-Less sensitive to salts-Lower PRACTICAL detection limits-Easier to interpret spectra(less multi-

ple charges)-Quick and easy-Higher mass detection-Higher Throughput(1000>samples per

hour)

MALDI/TOF Mass spec-trum

Re

lativ

e A

bu

nda

nce

m/z

0

10000

20000

30000

40000

50000 100000 150000 200000

(M+H)+

(M+2H)2+

(M+3H)3+

The Mass Analyzer: TOFTime Of Flight(TOF)

Flight TubeIon Source

Principle: If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will separate according to their mass to charge ratios.

20-25 kV

++

Calibration of the mass scale

The mass-to-charge ratio of an ion is proportional to the square of its time of flight in the analyzer (“drift time”).

2

22

L

Kt

z

m

t = Drift timeL = Drift lengthm = MassK = Kinetic energy of ionz = Number of charges on ion

The Mass Analyzer: TOF

Flight Tube

Detector

Ion Source

+

+ +

The Mass Analyzer: TOF

Flight Tube

Detector

Ion Source

+

++

The Mass Analyzer: Quadrupole

Quadrupole(Mass filter)-4 개의 molybdenum 막대로 이루어져 있으며 ,

한쌍 (1,2) 은 DC voltage, 다른 한쌍 (3,4) 은 Radio frequency voltage 가 가해진다 .

- 가해지는 전압의 진폭은 선택된 m/z 에 해당되는 ion 만 ion source 에서 detector 까지 통과하게 한다 .

- quadropole 의 전압을 바꾸면서 주어진 mass범위의 이온을 scanning 한다 .

The Mass Analyzer: Quadrupole

Detectors

+

-1000 V -100 V

Primary Ion from Flight Tube

L

D= 6-25 u

Ions are detected with a Microchannel Plate

+

-1000 V -100 V

L

D= 6-25 u

Ions are detected with a Microchannel Plate

+

-1000 V -100 V

e-

L

D= 6-25 u

Ions are detected with a Microchannel Plate

Multification by secondary emission

Secondary emissive materials: Beryllium oxide, magnesium oxide etc

+

-1000 V -100 V

e-

e- e-

e-

L

D= 6-25 u

Ions are detected with a Microchannel Plate

+

-1000 V -100 V

e-

e- e-

e-

L

D= 6-25 u

Ions are detected with a Microchannel Plate

+

-1000 V -100 V

e-

e- e-

e-

L

D= 6-25 u

e-

e-

e-

e-

e-

e-

e-

e-

~103

Amplification

Ions are detected with a Microchannel Plate

Tandem MS(MS/MS)

Tandem MS(MS/MS)

Tandem MS(MS/MS)

Laser

Sampleplate

Analytemolecules in matrix

Accelerationgrids

Drift tube Ion detector

Mass spectrum

Vacuum system

Vacuumlock

MALDI TOF MS

HiRes mass spectrum

Iondetector

MALDI ionsource

Ion reflector

The reflector focuses ion of same mass but different velocity on

detector; high resolution is obtained

Laser

High resolution TOF-MS with ion reflector

Daughter ion mass spectrum

Iondetector

Ion reflector

MALDI ionsource

CID

MS/MS spectrum of daughter ionsis measured in a single acquisition;

no pasting of segments;low sample consumption,

high speed, high sensitivity

LID

Laser

TOF/TOF-MS/MS with

Parent ionselector

Why interested in MALDI-TOF MS 분자량 측정

큰분자량 물질 분석

혼합물 분석 : 한 종류의 성분이 아닌 몇 종류가 혼재해 있어도 분석이 가능함

미량분석 : 매우 민감하여 미량의 시료도 분석 가능 함 : 펩타이드 경우 fmol 분석 가능

데이터 분석이 쉬움 : 분자 구조가 깨어 지지 않고 , 보통 다 전하를 (multiple charging) 띠지

않으므로 데이터가 다른 질량 분석기에서 보다 단순하여 해석이 용이함

염의 영향이 적음 : 생체단백질 분리에 이용되는 완충용액 , 염 등에 LC/MS 보다 영향을 덜 받음

분석이 신속함 : 시료와 Matrix 섞어 sample plate 에 떨어뜨려 용액을 말리는 시간 ( 약 5~10

분 ), MALDI-TOF 로 분석하는 시간 (1 분 이내 )

기기 사용 및 유지하기 위한 비용이 저렴 : LC/MS, GC/MS 처럼 질소 또는 아르곤 가스를 사용하지 않

고 , 미량의 Matrix 와 ACN, TFA 등을 이용함으로 시약 비용도 저렴함

Mass Spectrometry 분석-base peak-parent peak-radical cations-Isotopes

Peptide Sequencing

Biomolecule Analysis

* 과거에는 ?-Electrophoresis, chromatography, ul-

tracentrifugation-Not very precise*MS 이용하면 ?-Proteins, oligonucleotides, oligosac-

charides, lipids-Detect modifications and sequences

Peptide Mass Fingerprint-ing

• Analytical technique for protein identifi-cation (protein sequence)

• Unknown protein of interest cleaved into peptide by protease

• Collection of peptides resulting from this cleavage comprise a unique identifier of the unknown protein

• Mass measured with MALDI-TOF and ESI-TOF

• in silico compared to the genome

• Computer programs translate the known genome of the organism into proteins

• Theoretically cut the proteins into peptides with the same protease (ex.Trypsin: K or R)

• Calculate the absolute masses of the pep-tides from each protein

• the masses of the peptides of the unknown protein vs the theoretical peptide masses of each protein encoded in the genome

• Results statistically analyzed to find the best match

In Gel Digestion & Mass Spectrometry

Trypsin Digest

Cut out 2D-Gel Spot

Protein Peptides

Peptide Mass Fingerprinting

Trypsin

N KK

KK

KK

R

RR

RC

N

C

K

KK

K

K

K R

R

R

RProtein

Tryptic peptide mixture. Masses measured by MS. Every peptide has a basic C-terminus.

A protein can be identified in a database by matching masses of a subset of the tryptic peptides against calculated values.

MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS

PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW

EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK

SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE

SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL

LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE

PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK

GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED

HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT

intact protein

enzyme

peptide fragments

Peptide Mass Fingerprinting2D-Gel

In Gel Digestion

MS

848.11272.5492.6

883.22978.9

812.61432.33127.1996.8702.4164.92748.2

848.31272.7493.2882.62978.3364.1948.93128.8

Database

3514.22837.1263.9147.41429.7199.6142.3640.8

Is identical to

In Silico Digestion

“Spot removal”

Post Translational Modifica-tions(PTM’s)

• PTM’s are very important in signaling as well as metabolic pathways (e.g. phosphorylation)

• Often we want to know not only which modi-fication a protein has undergone, but exactly where in the sequence the modification lies.

• Many of the search engines allow for “vari-able” modifications, but very few at one time (combinatorialy explosive)

• There is great opportunity here for robust searches that find PTM’s reliably!

Protein sequence Analy-sis

For sequencing of an entire Protein…??Divide and Conquer !!!

Deduction of Full Amino Acid Sequence of a Proteinby Overlapping the Sequences Obtained from individual Peptides

Edman Degradation Sequentially Removes One Residue at a Timefrom the Amino End of a Peptide up to 50 times

Each round

can be complete

within 1 hr and

the Edman degradation

can be repeated

up to 50 cycles

in Practice.

Lymphomas and Leukemias

Regulatory Mutations

Chromosome 17

MessengerRNA

Her2 gene Her2 gene amplification

Overexpression

Her2 proteinHer2 protein

Normal expression