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Carbohydrates
Supplemental Slides
Biochem 5613
Figures: Lehninger Principles of Biochemistry 6th Ed., Nelson & Cox
Essentials of Biochemistry, 3rd Ed. OSU Custom Edition
Voet & Voet, Biochemistry 3rd Ed.
Voet, Voet & Pratt, Fundamentals of Biochemistry 2nd Ed.
Berg, Tymoczko, & Stryer, Biochemistry 2001

Glycoproteins
N-linked glycoproteins: Large, heterogeneous
oligosaccharides linked to asparagine side chains
O-linked glycoproteins: Smaller carbohydrates (usually
1-5 saccharides) linked to hydroxyl groups of Ser and/or
Thr side chains

What is wrong with

these structures?

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Glycoproteins
Carbohydrate may be a large amount of the glycoprotein mass
Carbohydrate is
hydrophilic; usually on
surface of a folded protein

Protein is dark grey;

carbohydrate groups are
in light grey
(a) Hemagglutinin
(b) CD2
(c) RNase B

Imperiali & OConnor, 1999

Glycoproteins
Oligosaccharide substituents of proteins are highly dynamic

RNase B protein is linked

to a heptasaccharide
substituent
Model displaying all
permitted structures
of oligosaccharide in
yellow and
superimposed on
protein core to
demonstrate freedom
of motion

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N-Linked Protein Glycosylation

Oligosaccharyl transferase (OST) is a 9 subunit, membrane

bound protein complex that covalently attaches a conserved
oligosaccharide to the protein substrate
The OST enzyme is required to activate the Asn amide to
allow it to serve as a good nucleophile. Why?

OST: Domain Structure

Hua et al (2008) Structure

The nine domains of OST serve different roles:

Stt3p: Catalytic domain

Stt3p, Ost1p, Ost2p, Wbp1, and Swp1p essential for function

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N-Linked Protein Glycosylation

N-linked glycosylation occurs in the endoplasmic reticulum
(ER) and is co-translational; may assist protein folding

N-Linked Protein Glycosylation

Transferred tetradecasaccharide is built up unit-by-unit by
glycotransferases (enzymes that transfer sugar groups
from diphosphate donors)

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N-Linked Protein Glycosylation

Transferred carbohydrate is a conserved oligosaccharide:
Man

Glc3Man9GlcNac2

Man
Man
Man
GlcNAc

GlcNAc

Man
Man

Man
Man

Man

In light grey: 5-sugar conserved

core of all N-linked glycoproteins

Glc
Glc
Glc

Maturation: Glycosidases and

glycotransferases work together
to modify the final glycan

N-Linked Glycosylation

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O-Linked Glycosylation
O-linked glycosylation of proteins occurs in the Golgi
apparatus
Sugar groups form a glycosidic bond with the side chain
hydroxyl group of Ser or Thr
One major purpose is immunological identification:
For example: Mucins are heavily glycosylated
proteins which are a significant component of mucus
Many cell-surface proteins are O-glycosylated

O-Linked Glycosylation
Core is not as conserved as in N-linked glycosylation

Gal-GalNAc-Ser/Thr
is the most commonly
observed core

However, other cores

are observed

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Protein Sequencing
Chapter 3.4
Biochem 5613
Figures: Lehninger Principles of Biochemistry 6th Ed., Nelson & Cox
Essentials of Biochemistry, 3rd Ed. OSU Custom Edition
Voet & Voet, Biochemistry 3rd Ed.
Voet, Voet & Pratt, Fundamentals of Biochemistry 2nd Ed.
Berg, Tymoczko, & Stryer, Biochemistry 2001

Amino Acid Analysis

Historically, protein content was determined by breaking the
protein into individual amino acids and quantitating the products:

Resuspend protein in 6 M HCl and boil overnight

Amino acid analysis provides information about content,

NOT the sequence

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AAA: Acid Hydrolysis

Complete conversion

Asn -> Asp

Gln -> Glu
HO

Ser, Thr
H3N+

COOH

Dehydrate

H3N+

COOH

Dehydroalanine
Partial degradation of Cys, Ser, Thr, and complete degradation
of Trp; do NOT get accurate numbers for these amino acids!

How do we determine a protein sequence?

1) How many chains?
Total amino acid content
N-terminal amino acid identification
2) Break protein down into peptide fragments
Disulfide bonds
Sequence-specific digestion
Chemical
Enzymatic (proteases)
3) Analyze peptide sequences
Edman degradation (Historically)
Mass spectrometry (Modern Method)
4) Combine all of this into a primary sequence

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Insulin
First sequenced protein (Sanger)
Two chains complicates N-terminal
identification
Three disulfide bonds

Protein Sequencing: Disulfides

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Protein Sequencing
Proteins are typically cleaved in multiple ways to allow the
generation of overlapping peptide fragments

Fragmentation of Proteins
Determining the sequence of very large proteins is complicated
It is common to break a protein into smaller peptide segments
to allow for easier sequencing

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Cyanogen Bromide
1)

Cleavage at methionine residues

3)

Methyl thiocyanate
leaving group

2)
Hydrolysis
4)

Protease Cleavage: Trypsin

Trypsin cleaves after lysine and arginine (red)

AA C-terminal to cleavage site can be anything except
proline (blue)

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Protease Cleavage: Chymotrypsin

Chymotrypsin cleaves peptide bonds C-terminal to large,
hydrophobic amino acid side chains
High rate of cleavage: C-terminal to Phe, Tyr, Trp
VERY slight non-specific cleavage at other hydrophobic
residues

Protease Terminology
Peptidase: breaks peptide bond
Exopeptidase: breaks peptide bond on a terminal amino acid
(exterior peptide bond)
Endopeptidase: breaks peptide bond somewhere in the
middle of (inside) the sequence
Carboxypeptidase:
Exopeptidase that hydrolyzes peptide bond at the C-terminus
(carboxyl terminus)
Aminopeptidase:
Exopeptidase that hydrolyzes peptide bond of the N-terminal
residue (amino terminus)

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Protease Specificities

Endo Lys-C
Endo Glu-C
Endo Asp-N
Name based on
specificity and
cleaved bond

Where would each of the following species cleave the

peptide below?
Endo Lys-C
Endo Glu-C
Endo Asp-N

Carboxypeptidase A
Trypsin
Aminopeptidase
Chymotrypsin
Cyanogen Bromide

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Peptide Sequencing
Edman Degradation

= Edmans reagent
Phenylisothiocyanate

Peptide Sequencing
Edman Degradation
Peptides can undergo multiple
cycles of Edman degradation to
identify entire sequence
Edman degradation can be
automated: protein sequencers
can carry out approximately one
round per hour
Up to 50 amino acids can be
sequenced fairly reliably before
small errors build up

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Modern Sequencing Methods:

Mass Spectrometry

All mass spectrometers have a common organization

Ion source

Mass analyzer

Detector

All separate based on mass to charge ratio

Electrospray Ionization (ESI)

Generates population of protein ions with many charge states

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ESI-MS: Mass reconstruction/deconvolution

Mass to charge
ratio observed
Positive charge is
generated by
protonation, so
add H+ for each
charge, each H+
adds 1 Da

Observed =

m+z
z

MALDI: Matrix Assisted Laser Desorption Ionization

Several different
matrices are
commonly used:

HCCA

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MALDI: Fewer Charge States

H4
11237

H2B H2A
13496 13951
H3(pT118)
15354

m/Z

Example: Mix of 4 proteins; observed [M+H]+, [M+2H]2+

ESI vs MALDI
Electrospray MS: Syringe full of protein/peptide solution
Ionize by concentrating charges in vacuum
Highly charged spectrum
Often connected to liquid chromatography systems
MALDI MS: Crystallize protein/peptide in matrix
Ionize using laser
Tend to see the parent mass
Simpler analysis of mixtures of ions

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Carboxypeptidase: Sequencing Exopeptidase

Great for sequencing based on remaining parent ion!

MALDI/Carboxypeptidase Sequencing

Time
series
Figure from John J. Lennon, ABRF News, 1997

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Tandem Mass Spectrometry for Sequencing

Any ionization technique

MALDI is common

MS/MS Sequencing

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