The Use of Spectroscopy and Chromatography in Proteomic Analysis

1. By
Shereen Mohamed Shehata
Supervised by
Dr. Mona AlShehri

2. The completion of sequencing the human genome belongs to the ground breaking
discoveries in life science during the past decade.
The accomplishment of the complete genome also brings along a new and more
challenging task for scientists: The Characterization of the Human Proteome.

3. • The term “proteome” was used first in 1994 and describes a set of all
proteins expressed by a given genome.
• A protein, the basic unit of a proteome, is a molecule composed of one or
more long chain amino acid residues, further forming secondary, tertiary,
and quaternary three dimensional structures.

4. • Proteomics, the main tool for proteome research, is a relatively new and
extremely dynamically evolving branch of science, focused on the
evaluation of gene expression at proteome level.
• Current proteomics deals with different issues, such as:
 Protein identification
 Quantification
 Characterization of posttranslational modification
 Structure and function elucidation
 Description of possible interactions

5. Approaches of
proteomics analysis
Sequence a few
peptide fragments and
match these sequences
with a previous mass
spectra libraries
De novo sequencing
when neither genomic
sequencing information
nor sufficient mass
spectrum data available

6. • The rapid development of proteomics during the past years was made
possible by progress in analytical instrumentation, especially in mass
spectrometry (MS).
• Beside the advances in technologies and methodologies dealing with
protein or peptide separation and sample complexity reduction, mainly in
liquid chromatography and electrophoretic techniques.

7. • The principle of chromatographic fractionation is based on the
interaction of the proteins or peptides with the stationary phase
and the mobile phase with instrumentation similar to that used in
conventional HPLC .
• The only difference is the magnitude of the flow rate (nl/min).
• The interaction may be:
 Adsorption on silica surfaces
 Partitioning on reversed-phase materials
 Ion exchange
 Affinity
 Size exclusion

8. • The ever-increasing demand for more selectivity, sensitivity and
specificity has led to three main developments:
 Improvements in existing RP hydrophobic stationary phases, allowing
operation with very low or no trifluoroacetic acid (TFA)
 The emergence of monolithic columns, whose separation medium consists of
a continuous, rigid polymeric rod with a porous structure, enabling faster
separations
 The development of miniaturized columns in a chip format

9. A multidimensional (i.e., 2D or 3D) method that combines separation
methods with different separation mechanisms with the following
advantages:
 Significantly improve the chances of resolving a complex mixture of proteins
into its individual constituents
 Extensive fractionation lowers the dynamic range requirements on the
instrumental technology (i.e., MS) used to detect the individual protein or
peptide species
 It maximizes the overall peak capacity

10. • Mass spectrometers consist of three basic components:
 An ion source
 A mass analyzer
 An ion detector
• It requires a method to transfer molecules from solution or
solid phase into an ionized gaseous phase either by:
 Matrix assisted laser desorption/ionization (MALDI)
 Electrospray ionization (ESI)

12.  It has made ESI more convenient for solid sample analysis
 Large biomolecules have also been successfully detected
Ambient desorption electrospray ionization (DESI)

13. • After ionization, the sample reaches the mass analyzer, which separates
ions by their mass-to-charge (m/z) ratios. Ion motion in the mass analyzer
can be manipulated by electric or magnetic fields to direct ions to a
detector.
• Four basic types:
 Time-of-flight (TOF)
 Ion trap
 Quadrupole (Q)
 Fourier transform ion cyclotron resonance (FTICR)

14. • All four types differ in:
 Sensitivity
 Resolution
 Mass accuracy
 The possibility to fragment peptide ions
• Hybrid TOF/TOF, Q/TOF and ion trap/FTICR instruments can be also used
resulting in fragment ion spectra which are often more extensive and
informative and combining the advantages of each technique.

15. • This technique based on spectral counting or the comparison of signal
intensities across samples in a narrow m/z range.
• There are two types of quantitation:
 Label-free quantitation
 Label-based quantitation

16. • The basis of this approach is that the peak height or the area of a peak at a
selected mass-to-charge ratio is obtained by counting the number of ions
forming this peak (i.e. Spectral counting)
• The disadvantage is that the more abundant proteins can mask the
proteins of low abundance.

17. Multiple reactions monitoring (MRM) vs MS/MS operating mode

18. Allowing equivalent peptides (or
peptide fragments) to be identified by a
specific increase in mass.

19. Several labeling techniques can be used, which are summarized in this table:

21. 1. Titanium columns for selective enrichment of phosphopeptides
2. Immobilized metal-ion affinity chromatography (IMAC) of phosphorylated
peptides
3. Ion exchange chromatography (IEX) - RP HPLC
4. Monolithic columns for analysis of proteomic samples
5. Miniaturization
6. Ultra high pressure liquid chromatography (UPLC)
7. Liquid chromatography-Capillary electrophoresis (LC-CE)

22. Enrichment followed by subsequent separation on RP separation column or nano-LC
system.

23. • Phosphorylated proteins and peptides show a high affinity towards metal ions
and form quite stable complexes with these ions.
• The most frequently used ions are Fe3+, Ga2+, Ni2+, Cr2+, Al3+, Zn2+ and Cu2+.
Metal cations are chelated by a multidentate ligand, which is immobilized onto
a support material.

24. • Unfortunately, it is not only the phosphopeptides which bind on to such a
column, but also the acidic non-phosphopeptides.
• In order to minimize this nonspecific binding, all free carboxyl groups of
the peptides in solution have been converted to the corresponding methyl
esters and then analyzed with IMAC followed by nano HPLC-MS.

26. Advantages of monolithic columns in comparison to conventional HPLC
columns:
 Easier preparation and functionalization
 Enhanced mass transfer
 Higher column efficiency
 More robust

27. However, monolithic columns suffer from one very important limitation:
They are very often and very easily overloaded.
One possible way to resolve this problem is to use serial linkage of these
columns.

28. • Monolithic columns can allow the use of online enzymatic reactors.
• Trypsin has been immobilized on a monolithic column and compared to
the off-line in-solution digestion.
• The offline digestion took about 12 h, whereas a digestion of comparable
efficiency on the monolithic column took about 30 s.
• This approach needs further tuning but it seems to be promising in terms
of increasing the sample throughput.

29. • Another important aspect of future development is the miniaturization of
instrumentation, primarily of the separation columns.
• There are many publications describing miniaturized chips and their use
for separation of proteins and peptides.
• These chips suffered from high dead volumes and high chemical
background in MS, originating from the glue for the tips.
• Furthermore, the majority of chip developments focused on
electrophoresis.

30. • The development of a new chip designed to use existing nano-HPLC and
MS hardware and the use of a nano-HPLC chip for RP and 2D separation of
peptides obtained through tryptic digest of rat plasma were reported.

31. • Decreasing the particle size of the stationary phase by a factor of 2, from 3
to 1.5 µm, would increase the backpressure 4-fold (pressure is inversely
proportional to the square of the particle size).
• This would improve the peak shape and increase the column resolution.
• The use of UPLC for functional genomic discrimination of metabolic
phenotypes has been reported.
• The results achieved with UPLC were significantly better in comparison to
the results achieved with conventional HPLC.

32. Advantages of HPLC and CE multidimensional procedures:
 They can be automated
 Sensitive
 Reproducible
 Fast
 Can be used for quantitative work

33. • The on-line LC-CE-ESI-FTICR-MS was employed to study the bovine serum
albumin (BSA) tryptic digestion product.
• Low detection limits were found (low ng/ml), while a high sequence
coverage (93 %) was obtained using this multidimensional set-up.
• An LC-CE-MS coupling was also used to separate peptides enabling protein
identification in complex mixtures.

34. The on-line LC-CE-ESI-FTICR-MS

35. • A similar strategy has been used for the analysis of human saliva and
mouse brain mitochondrial proteome.
• The strategy consisted of an off-line coupling of CE to nano-RP LC-ESI-MS
via a fraction collector.
• CE enables in-capillary sample concentration for the use of larger volume
injections.
• The developed CE-LC-MS method:
 Resulted in complementary data
 Resulted in higher number of detected mouse mitochondrial peptides and
proteins

36. Modern separation methods (LC and CE) have been used in a multidimensional format,
LC/LC or LC-CE for the separation of proteins/peptides combined with MS for protein
identification and profiling.

37. • MS-based proteomics by the rapid development of mass analyzers with
higher mass resolution and superior sequencing capabilities, along with
separation systems has established itself as the leading technology for a
high-throughput qualitative and quantitative analysis of protein mixtures.
• However, the analysis of difficult types of proteins that are present at low
abundance, hydrophobic, or extensively modified, remains to be
challenging and will require further development of improved analytical
tools and methodologies.