The Proteome of a living organism is the collection of proteins that are present in that organism. All these proteins are encoded based on the genome of an organism, but undergo substantial differentiation (Post Translational Modifications or PTMs) after translation. In the human DNA there are about 30.000 genes, but the number of human proteins is estimated to be around 300.000 different types.

In addition to the huge complexity of the proteome composition, proteins levels vary over a range of 109 orders of magnitude! Human plasma contains high abundant proteins such as Albumin (55% in plasma) and Immunoglobulins, but the proteins of interest (e.g. Interleukins or tissue leakage markers) are low abundant and a million to a billion times lower in concentration. On the human scale, this abundance difference relates to searching for a small blade of grass in the complete Amazon rain forest.

The structure, modifications and levels of these proteins reflect the physiological state of an organism. Proteomics is the field of research that has set out to map all the content and dynamics in the proteome; especially the human proteome.

Proteomics can be divided roughly into four areas which can blend into each other:

• Targeted proteomics
• Structural proteomics
• Biomarker validation
• Biomarker discovery

A large portion of the effort in Proteomics research is done in the area of biomarker research. Scientists are comparing healthy states and disease states of an organism to identify differences in protein expression. Depending on the disease a protein can be found to be up- or down regulated. When this difference in expression is observed, a potential biomarker has been discovered.

The next step would be to verify the validity of a potential biomarker by scanning a larger sample pool specifically for that marker. This typically involves two stages in biomarker research:

• The discovery phase, comprehensive approach analyzing few samples in depth
• The validation phase, targeted approach analyzing many sample for the presence of specific proteins

Not all proteomics research is related to biomarkers; unraveling a disease pathway requires a more targeted approach. Post translational modifications play a critical role in cellular processes and usually involve the addition of a small molecule to the protein that is critical to the functionality of the protein. Fortunately the PTM’s often introduce a special feature that can be exploited in the separation and detection phase.

For practical reasons > 90% of proteomics research is centered around peptide detection (so called Bottom-Up approaches), but preferably the digestion step is excluded from the experiment. More and more effort in proteomics is being spent on intact proteins analysis or so called Top-Down approaches.

Sources:
Anderson and Anderson – Mol Cell Prot - 2002 - 11 - 845-869

The enormous diversity of proteins in the proteome places a high demand on analytical techniques. Typical requirements are:

• High Sensitivity to allow detection of femtomoles or even lower.
• Increased Resolution and Selectivity for more reliable identification.
• Maximum Robustness and Repeatability for consistent results at a global level.

Sensitivity – NanoLC

Dionex has a pioneering role in the field of nano HPLC – a technique that generally involves the application of columns with an internal diameter of 75 µm. Use of these extremely narrow columns typically increases the sensitivity about 4000 times. In addition these columns are operated at low flow rates of around 300 nl/min and can be seamlessly integrated with mass spectrometry.

Resolution and Selectivity

The separation demand in proteomics is higher than ever before in science. The almost infinitely complex samples that are being analyzed often need multiple steps in the separation strategy. LC provides the most extensive capabilitiesand usually is the final separation step before MS identification.

Structural Proteomics
After sample extraction and purification the remaining sample is often separated at the protein level. Separation of intact proteins can be a valuable approach when combined with Fractionation in preparation for Bottom-Up approaches. High resolution intact protein separation columns can also offer the necessary separation power in a Top-Down strategy in Structural Proteomics.

Biomarker Discovery
Biomarker discovery requires a comprehensive analysis of a limited number of samples. It often involves comparing a diseased state with a healthy state to find up- or down regulated proteins. Because one searches for unknowns, high separation power is demanded here e.g. in the form of 2D separation methods. The limited sample number at this stage allows the utilization of a longer separation method.

Biomarker Validation
Biomarker validation is increasingly performed as a result of the many potential biomarkers that have been discovered so far. The potential biomarkers from the discovery phase are now validated using a much larger sample set. A higher throughput and excellent repeatability are required. The targeted nature (one knows what to look for) allows a less extensive separation typically in the form of 1-D separations. Combined with targeted MS detection, this allows for a higher throughput and excellent repeatability.

Targeted Proteomics
Sometimes the approach to the protein of interest cannot be as straightforward as above. Investigations of e.g. disease pathways require a targeted approach and often involving PTM differences. In PTM analysis a special subset is isolated by affinity based separation step, e.g. TiO₂ for phosphorylations. Targeted Proteomics covers the field of targeted approaches based on protein features.

Robustness and Repeatability

In proteomics the samples always require sample preparation and are always analyzed by mass spectrometry. The limited sample starting amounts, the multistep analyses, and the operating cost of the expensive MS equipment necessitate a reliable LC system to perform the chosen separation strategy. The advanced wellness features of the UltiMate 3000 platform provide early warnings to allow planned preventive maintenance resulting in improved system robustness.

Excellent hardware and automation are key to repeatability in a separation strategy. UltiFlow technology in the Nano LC instruments and applications such as the Automated Off-Line 2-D LC provide this excellent repeatability by working with minimal user interference.

UV detection cannot be used to identify the peptides and proteins, but functions as an excellent tool to monitor and optimize the qualitative and quantitative aspects of the analysis.

Sample Identification

Proteins and peptides are composed of amino acids that are linked together in chains. Only 20 different amino acids occur naturally, but these 20 building blocks allow an infinite number of combinations. The way to identify the proteins or peptides is done by sequencing the order in which the building blocks are linked together. Mass spectrometry is the tool to perform this job.

Before a sample can be analyzed by mass spectrometry it first needs to be ionized in such a way that the molecule exiting the LC system remains intact. Of the various ionization techniques two are most commonly applied in proteomics because of their ability to ionize large molecules without breaking them down to smaller elements. These so called soft ionization techniques are:

• Electrospray ionization or ESI
• Matrix Assisted Laser Desorption Ionization or MALDI

Electrospray

The electrospray process was developed by John Fenn (Nobel Prize 2002) and allows online coupling of nanoLC to the mass spectrometer. In the ionization process the LC effluent exits a charged needle in the MS ion source as a spray. During the transfer from needle exit to MS inlet, the droplets shrink by an evaporation process and the charge (H⁺) is transferred to the peptides. The resultant gas phase ions can be analyzed by the mass spectrometer.

Due to the direct coupling to the LC, ESI is also referred to as an online ionization process and puts constraints on the chosen LC method. The gradient elution should be performed with volatile solvents and low ion pairing conditions. In 2D methods either volatile salts are used or a desalting step is required.

Formic Acid (FA) is preferred from an ionization point as it exhibits lower ion suppression than TrifluoroAcetic Acid (TFA). However Reversed Phase chromatography often proves less efficient with a weaker ion pairing agent. This can be countered through the use of Dionex PepMap nanoLC columns as these allow the use of Formic Acid as an RP ion pairing agent without compromising on separation performance.

MALDI

MALDI was developed over several years by different research groups. Franz Hillenkamp and Michael Karas initiated MALDI with small peptides and Koichi Tanaka (Nobel Prize 2002) was the first to ionize proteins by MALDI.

MALDI is an offline ionization process, whereby the LC effluent is “spotted” on a target in the presence of an organic salt - the “matrix”. The spotting usually occurs at timed intervals and with simultaneous addition of the matrix using e.g. the Probot. The matrix crystallizes on the MALDI target upon solvent evaporation, encapsulating the sample.

After placing the spotted MALDI target in the MS, a laser shoots on the spot. Matrix and laser wavelength have been chosen to maximize energy transfer, resulting in an evaporating plume. In this plume the matrix molecules transfer the H+ charge to the sample molecules, which are then analyzed by the MS. 

Due to the indirect coupling to the LC, MALDI allows certain benefits. The LC separation is frozen in time allowing extended time for MS analysis. Also a sample can be spotted in one location and analyzed on another, allowing efficient use of capital investment in MS equipment. In addition MALDI is also relatively insensitive to the presence of nonvolatile additives in the LC mobile phases.

In practice ESI and MALDI are complementary techniques and many publications show that sample coverage can be maximized when results obtained with both techniques for one sample are combined.

Detection

After the peptides have been ionized they enter the mass spectrometer where they are separated based on their mass-to-charge-ratio (m/z). Typically the peptides are also sequenced by fragmenting an isolated m/z value. The combination of peptide mass and fragment masses are then analyzed by database search algorithms to yield a peptide and protein identification.