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BioMedChem M3 > 22/23 - Fluorescence, NMR, MS > Flashcards

22/23 - Fluorescence, NMR, MS Flashcards

1
Q

Non-Naturally occuring GFP Analogs

A

Obtained through Mutagenesis / Synthetic modification

Diversity covers nearly the entire visible spectrum
Serius / BFP / CFP / YFT etc

Conjugation system –> FLUORESENCE
small changes that affect the conjugation –> various colors

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2
Q

FP = Fluoresence Proteins

in PROTEIN LABELING experiments

A

FP –> either C-terminus or the N-terminus of the target protein
sometimes BOTH are needed for function so:
FP -> inserted inside the protein chain

Gly-linker, common but not mandatory
offers flexibility/avoids steric conflicts

  • *Expression levels MUST be monitored** to ensure that the protein
  • *Still functions normally!**
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3
Q

Uses of GFP-type Imaging Experiments

A

HeLa Cells

multicolor labeling of the cells via confocal microscopy

Show where EACH PROTEIN is LOCALIZED

mitochondria / actin / histone / golgi / etc

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4
Q

Uses of IN VIVO Labeling using FPs

A

help visualize cell types in
whole animals / organs / tissues / cell cultures

Neurobiology / immunology / development

Transplantology / Carcinogenesis

Real-Time Monitoring of CELL CYCLE PROGRESSION
of living tissues

G1 Phase / S /G2 / M phases

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5
Q

LIMITATIONS of IN VIVO labeling using FPs

A

LIGHT ABSORPTION BY:

MELANIN

HEMOGLOBIN
has extensive conjugation –> absorbs some light

Optimal optical window for living tissues is:
650-700nm & 1,100nm

which is the “near IR” region = 700->1,700nm

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6
Q

Uses for NEAR IR PROBES

“NIR”

A

Visualization of ANATOMICAL FEATURES
image guided surgical removal of disease tissues
Blood+lymph vessels / GI tract / Bile Duct / Uterus

  • *Biomedical Imaging**
  • *high imaging resolution** with increasing tissue penetration depths

NIR-1 = 700-900nm & NIR-2 = 1,000-1,700nm
probes allow imaging of inorganic/organic macromolecules

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7
Q

Give 2 Reasons why FLUORESCENT PROBES are USEFUL

A
  • *FPs encoded in proteins of interest** make it possible to observe their:
  • *LOCALIZATION / MOVEMENT / TURNOVER**
  • *FPs targeted to cells** enable visualization of:
  • *MORPHOLOGY / MOVEMENT**

useful for studying disease states

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8
Q

What light spectrum does NMR measure?

A

RADIO WAVES

measure the

NUCLEAR SPIN

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9
Q

NMR Signals are the result of what?

A

Absorption of ELECTROMAGNETIC RADIATION (resonance)

by NUCLEI

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10
Q

NMR Steps

A
  • Spectrometer
    • gives of an electromagnetic pulse
    • the sample absorbs this energy
  • FID = Free Induction Decay
    • electromagnetic radiation emission by each excited nuclei
  • Fourier Transform
    • digests the data / MATH
  • SPECTRUM
    • Representaion of nuclei in the molecule
    • each nuclei emitted radiation at a DIFFERENT FREQUENCY
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11
Q

NMR Specrum is what?

A

Representation of the NUCLEI in the molecule

Each nucleus emitted radiation at a

Slightly different FREQUENCY

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12
Q

What can we measure and NOT measure with NMR?

A

NMR = Radiowaves that measure NUCLEAR SPIN
only a select few nuclei are good canidates for NMR:

NMR Nuclei
1H (99.9%), 13C (1.1%)
14N, 17O, 19F​

elements that _LACK A NUCLEAR SPIN_ will _NEVER exhibit an NMR phenomenon_

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13
Q

What type of SOLVENTS do we use for NMR? and WHY?

A

DEUTERATED SOLVENTS
CD3OD / CDCl3

Deuterates = like 2H
are INVISIBILE in NMR experiments,
does NOT interfere with the signal

Our goal is to have:
minimal interference from the solvent

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14
Q

Differentiating SIGNAL in NMR

A

Nuclei have DIFFERENT electronic environments
Emit a DIFFERENT TYPE OF ENERGY

As a result these nucleu have resonance @ different FREQUENCIES
= X-axis of spectrum

  • *Electrons** (electron density) SHIELD each nucleus from the
  • *Applied Magnetic Field**
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15
Q

Downfield = ?

A

DE-Shielded

Further LEFT,
on the X-Axis (chemical shift or frequency, PPM or Hz)

GREATER Frequency

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16
Q

Upfield = ?

A

SHIELDED

Further RIGHT–>,
​ on the X-Axis (chemical shift or frequency, PPM or Hz)

lower Frequency

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17
Q

Chemical Shift (δH) INCREASES As……

A

ELECTRONEGATIVITY of adjacent atoms INCREASES

Chemical shift δH = position on the x-axis of the spectrum
protons in different electronic environments exhibit
Different Chemical Shifts

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18
Q

Chemical Shift δH​

A

Value of a RESONANCE on the X-Axis of the Spectrum

Each electron environment causes a characteristic δH​
which allows for us to predict the nature of the environment/type of the signal of interest (proton)

δH​ INCREASES as ELECTRONEGATIVITY of adjacent atoms INCREASE

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19
Q

Why is a hydrogen adjacent to an oxygen is more deshielded vs a hydrogen next to a carbon?

A

OXYGEN IS MORE

ELECTRONEGATIVE

The hydrogen (proton) is being “deshielded” by the Oxygen which is pulling electrons away

Functional groups such as -OH de-shield protons

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20
Q

Why is the CH2 adjacent to the ARYL GROUP

more SHIELDED

than a normal CH2 group?

A

the ARYL Group is a

CONJUGATED SYSTEM / AROMATIC GROUP

that SHIELDS the proton

The hydrogen (proton) is being shielded by the conjugated system which is contributing to the degree of electrons round the proton

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21
Q

What is INTEGRATION?

in terms of Spectral Analysis

A

The AREA UNDERNEATH A PEAK in a NMR spectrum

correlates directly with the number of nuclei involved

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22
Q

When is H NMR NOT HELPFUL in determining structures?

A

ABSENCE OF HYDROGEN ATOMS

a lot of CONJUGATION –> have less hydrogen atoms

lead to blind spots in the structure

so we would then use C-NMR

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23
Q

Negatives to 13C NMR experiments

+ Positives

A

13C is only 1.1% in relative abundance, not-abundant

LONGER EXPERIMENT TIMES

H1 is much faster because there is much more of them 99+%

  • *13C NMR** has a LARGER range for X-axis Chemical Shifts
  • *δC: 0 – 220 ppm**

appear as singlets

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24
Q

Types of NMR Methods

A

COSY H-H

NOESY / TOCSY = 2D H-H
2D Method = Box Plot

HSQC = H-C

HMBC = H-C

25
Q

COSY

NMR Method

A
  • *H-H**
  • *COrrelation SpectroscopY**

HOMOnuclear Spin Coupling

Corralates hydrogens next to hydrogens.

Off-diagonal contours indicate coupling
proton-proton coupling of H-Groups

SHOW BONDS / DIRECT CONNECTIVITY

26
Q

What NMR Method is useful in determining

Similar H-Groups next to the “same” H-group?

Ex. CH2-CH2 or CH=CH

A

COSY

H-H

Proton-Proton Coupling of adjacent H-Groups

27
Q

HSQC

NMR Method

A

Heteronuclear Single Quantum Coherence

DIRECT H-13C heteronuclear spin coupling

Will ONLY show H’s that connect to C’s = CONTOUR
H on X axis
C on Y axis

matching will show that those two are connected
does not show quartenary carbons, or resonances due to -OH or -NH

28
Q

Which NMR Method tells us

Which H is directly connected to which C?

A

HSQC

“H->C”

DIRECT heteronuclear spin coupling

29
Q

HMBC

NMR method

A
  • *H**eteronuclear Multiple Bond Correlation
  • *H-C** Heteronuclear spin coupling
  • like HCQC but shows MORE INFO*

2-3 Bond Correlations Observed
Correlation BETWEEN each structure
key to PIECING the structural fragments together

typically used after the other tests

30
Q

Which NMR method shows us

the correlation BETWEEN each structure?

observes 2-3 bond correlations

A

HMBC

H-C

Shows more info than HCQC, typically used after the other tests

31
Q

NOESY

NMR method

A

Nuclear Overhauser Enhancement SpectroscopY

H-H homonuclear spin coupling
DIPOLE DIPOLE THROUGH-SPACE INTERACTIONS
does NOT read through bond interactions
MAX 5Angstrom distance

Shows where things are pointing in space + Shape
Stereochemistry
Cis vs Trans
E vs Z

32
Q

Which NMR method shows us

Stereochemistry (shape) of the sample and (stereo)Configuration of double bonds?

Cis / Trans

E / Z

A

NOESY

H-H

shows THROUGH SPACE PROXIMITY

33
Q

MRI Facts

Magnetic Resonance Imaging

A
  • *Same principles as NMR,**
  • except we use the WHOLE BODY (Tube)*

We DONT simply “image” the whole body at once
signal would be too messy, must be fine tuned

Superconducting MAGNET

data collection produce high quality images

34
Q

Mass Spectrometry Steps

A
  1. Sample IONIZATION
    1. shoots ENERGY
  2. Sample Breaks apart / separation
    1. forms FRAGMENTS
  3. ​DETECTION of these ions & fragments

degrative process

“Reverse Engineering”

35
Q

Mass Spectrometry Definition

A

Any technique to achieve the introduction of a sample

followed by Ionization -> Seperation -> detection of ions

Destructive method,
allows you to observe how a metabolite fragments

36
Q

High Resolution MS

A

Allows you to determine MOLECULAR WEIGHT

down to MORE decimals so that we can determine

More EXACT Molecular Formula

37
Q

Molecular Ion Definition

MS

A

The ION obtained from the

LOSS of an electron

from a molecule M+

38
Q

Base Peak Definition

​MS

A

the MOST INTENSE peak in the MS

39
Q

Fragment Ions Definition

​MS

A

LIGHTER IONS fromed by the

decomposition of the molecular ion

40
Q

Pseudo molecular ions, or adducts

MS

A

[M+H]+,

[M+Na]+,

[M-H]-, etc.

41
Q

Obtaining STRUCTURAL INFORMATION from MS

GRAPH, what is Y & X?

A

Y = Relative Abundance

X = m/z , mass to charge ratio of fragment

SIMPLY A MATH PROBLEM

To identify fragment ions in a spectrum, determine the difference between the

[m/z value of a given fragment ion] - [that of the molecular ion]

42
Q

Mass Spectrometry what bonds break and in what preference?

A

WEAK BONDS > strong bonds
weak bonds break more easily

Bonds that break to form MORE STABLE FRAGMENTS
break in prefrence to those that form less stable fragments

43
Q

Types of IONIZATION SOURCES

A

EI = Electric Ionization
HARD ionization process

CI = Chemical Ionization
soft ionization process

  • *MALDI =** Matrix-Assisted laser desorption ionization
  • soft ionization w/ assist of matrix*

ESI = Electrospray ionization
coupled to HPLC, Liquid sample passed through HIGH VOLTAGE

DESI = Desorption electrospray ionization
analysis DIRECTLY off surface, quick and fast.

44
Q

Which type of ionization technique is a

HARD ionization process & what is a hard ionization process?

A

ELECTRONIC IONIZATION = EI

much EXCESS energy and possibility of
extensive fragmentation

that gives us Fingerprint Information

vaporized compound, we sometimes want this

45
Q

Which type of ionization technique is a

SOFT ionization process​ & what is a soft ionization process?

A

MALDI is also a “soft” ionization

CHEMICAL IONIZATION = CI

less energy,
molecular ion predominates, NOT VERY FRAGMENTED

has to be a volatile compound

provides molecular MASS information / MW

46
Q

MALDI

type of Ionization Technique

A

Matrix-Assisted Laser Desorption Ionization

The MATRIX ABSORBS/ASSISTS large amounts of energy
and transfers some of theis energy to the substrate

to MINIMIZE substrate DECOMPOSITION

SOFT IONIZATION
substrate does not decompose much due to the MATRIX

47
Q

ESI

type of ionization technique

A

Electrospray Ionization

liquid sample is passed through a HIGH VOLTAGE metal capillary

COUPLED TO HPLC

nebulized & carried by Nitrogen Gas
to produce an aerosol of charged droplets

48
Q

DESI

type of ionization technique

A

Desorption Electrospray Ionization

combines real-time microscale solvent etraction in
fast movign droplets with ESI

MS analysis DIRECTLY OFF OF SURFACES

quick & direct!

49
Q

Types of Mass Analyzers

MS

A

Magnetic Sector Analyzer
uses a magnetic field, different size = different trajectory

Quadrupole Analyzer
seperated by passing through a MASS FILTER, different size = diff detection

Time-of-flight (TOF) Analyzer
ions are seperated according to their VELOCITIES

50
Q

What do MASS Analyzers do?

A

After ions are generated they must be SEPERATED & DETECTED

Based on mass to charge ratio = m/z
ions behave differently in ELECTRIC or MAGNETIC fields

51
Q

Magnetic Sector Analyzer

type of Mass Analyzer for MS

A

New ions are passed through a MAGNETIC FIELD
the radius of curvature : m/z

allolows for distinction between HEAVY & LIGHTER IONS
VARY IN TRAJECTORY

Different size = different trajectory

52
Q

Quadrupole Analyzer

type of Mass Analyzer for MS

A

Ions are seperated by passing them through a
quadrupole mass filter = 4 poles/parallel rods

that create a ELECTRIC FIELD around the ion’s flight path

Different Size = Different detection

53
Q

Time-of-Flight Analyzer

TOF

type of Mass Analyzer for MS

A

Ions are seperaed according to their
VELOCITIES
generated in a short burst –> then accelerated

adv = speed of detection / sensitivity / unlimited mass range

  • *Time taken to traverse a SPECIFIC PATH** depends on the
  • *MASS of the ion**

typically employed with MALDI

54
Q

What is USELESS to detect in COSY?

A

Only shows H-H bonding next to similar Bonds

Does not show what H-C connectivity

55
Q

What is USELESS for HSQC?

A

Quaternary Carbons
Resonances due to -OH or -NH
>1 bonds away

Showing H-H connectivity
Stereochemistry / configuration

Only tells us which H is DIRECTLY connected to WHICH C

Heteronuclear Single Quantum Coherence
DIRECT H-C, 1* Bonds

56
Q

What is USELESS for HMBC?

A
  • *Initial identification,**
  • mainly for piecing the fragments together*

H-H bonds or 1* bond correlations

Heteronuclear Multiple Bond Correlation
H-C heteronuclear spin coupling

for 2-3 bond correlations, further away

57
Q

What is USELESS for NOESY

A

does not read through bond interactions
only goo for through SPACE interactions

Nuclear Overhauser Enhancement SpectroscopY
H-H for stereochemistry / config of double=bonds / stereoconfiguration

58
Q

Imaging Mass Spectrometry

IMS

A

Provides a 2-3 Dimensional visualization of the distribution of chemicals from biological samples

Spatial mapping of molecules over a phenotype

for MOLECULAR IMAGES

allows for
distinction of different molecular species
like B-amyloid Plaques in ALZHEIMERS

BioMedChem M3 (8 decks)

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