KEY POINTS

In general, workflow strategies in proteome research can be divided into top-down and bottom-up approaches.^6,7 Top-down strategies refer to all methods and proteome workflows, which use intact proteins for a first separation or fractionation step. Consequently, all gel-based, screening (protein profiling) as well as liquid chromatography (LC)-based methods of intact proteins have to be considered in this context.^8,9 On the other hand, bottom-up strategies investigate proteins by analysis of their respective peptides, involving the enzymatic digestion of the whole samples to obtain smaller peptide fragments that are still sufficiently distinctive for protein identification.^10,11

New bioanalytical methods based on mass spectrometry could provide diagnostic and prognostic information for cancer and other disease-related biological fluids. However, without a separation process beforehand, mass systems have continued to be limited in their ability to analyse and identify potential markers. A major reason is the extremely high complexity of protein samples with a dynamic range up to 1012. Blood serum seems to be an ideal clinical sample for proteomic analysis because it can be easily collected from the patient and has a very high protein concentration (60–80 mg/mL). Nevertheless, only 22 proteins make up 99% of the whole serum proteome and many potential biomarkers are likely to be present at lower protein concentrations.¹⁴ Therefore, several sophisticated approaches such as LC-electrospray ionization (ESI) MS¹⁵ have been developed as sensitive detection tools to resolve and visualize complex peptide and protein mixtures over a broad mass range. In general, LC–MS methods generate large and highly complex data sets, which require powerful algorithms and software tools to handle and analyse them.¹⁶ Furthermore, LC–MS strategies are not yet very suitable for screening a large numbers of samples due to time-consuming analysis. However, screening by profiling methods enables the analysis of numerous samples in a short period of time and can therefore be employed for mining out differences between a huge number of deceased and healthy samples. Moreover, profiling methods allow the simultaneous measurement of a range of markers that result in, statistically, more stringent differentiation and a better classification of patient groups.¹⁷ For example, mass fingerprinting of blood samples can lead to some disease markers because the leakage of peptides and proteins into the bloodstream by cells, in response to a disease, can be used as markers.^18,19

In this review, we want to provide an overview about different profiling strategies based on a new approach termed as material-enhanced laser desorption/ionization (MELDI).²⁰ MELDI has been introduced as a mass spectrometric-based technique for pattern analysis of biological samples. While being similar to other approaches such as surface-enhanced laser desorption/ionization (SELDI) ²¹ or protein profiling by magnetic beads,²² MELDI enables both screening of biological samples, as well as identification of serum constituents in conjunction with LC–MS.

Sample Collection, Handling and Storage

Several studies have examined the effects of pre-analytical procedures including protein immobilization,²³ matrix application²⁴ and sample processing²⁵ by determining their relative influence on the results. With the extra sensitivity of MS, it is likely that even quite small changes during sample collection and storage, particularly those leading to degradation and breakdown into smaller fragments, might be detected. In many cases biological fluids like serum, plasma or urine are investigated to monitor diseases at their different stages, because they might be rich sources of potential disease markers that could reflect the ongoing physiologic state of an individual organism.²⁶ Blood serum especially, is used for many analytical approaches because it contains a total of 60–80 mg/mL of protein with a concentration range spanning up to twelve orders of magnitude. As blood perfuses tissues and organs, proteins and many other compounds, which are secreted or lost from damaged or dying cells, are released into the blood circulation after cleavage processes into smaller protein fragments.²⁷ Many researchers believe that an effective characterization of potential disease markers will require methods to remove the most abundant proteins prior to analysis.

However, the reproducibility and effectiveness of many depletion methods have always been questioned and there are serious concerns that potentially important markers which are associated with high abundant carrier proteins (e.g., albumin) could be removed by depleting them.²⁸ Moreover extensive sample handling during the depletion process increases the chance of sample loss, protein degradation and modification artifacts, resulting in substantial sample-to-sample variation. Pre-analytical aspects, such as centrifugation (speed, time and temperature), storage time and temperature, exposure to freeze–thaw cycles are also likely to be important, and require further investigation or, at least, consistency within studies. An interesting observation was made by Villanueva et al.,¹⁹ who provided evidence that disease markers might be generated after the patient's blood collection ex vivo as a result of proteinase-mediated enzymatic cleavage. The impact of these results in biomarker discovery is quite significant as it is widely believed that ex vivo proteolysis should be suppressed because it destroys endogenous biomarkers. Therefore the authors recommend that after blood sampling, disease-specific proteinases should not be suppressed by the addition of proteinase inhibitors as it might prevent the generation of disease markers.

The most common variations between laboratories and/or medical institutes include blood-sample tubes and other preparation aspects.²⁹ There are studies examining whether different types of blood-collection tubes add molecules to specimens that might appear as interfering or confounding peaks during MS profiling.³⁰ Drake et al.,³¹ concluded that most types of commonly used blood-collection tubes for serum collection add polymeric components that can be detected by mass spectrometry covering the m/z range of 1000–3000. These peaks potentially complicate and compromise the interpretation of profiles in the low-molecular-mass range. Very often silicones are used as lubricants for stoppers or coatings for the internal surface of tubes, and can confound peaks, as well as the polymeric surfactants such as polyvinylpyrrolidones or polyethylene glycols, which might be added to influence surface wetting.

Surface-enhanced Laser Desorption/Ionization Mass Spectrometry (SELDI)

Surface-enhanced laser desorption/ionization (SELDI), which was originally introduced by Hutchens and Yip in 1993,³² has proven to be an effective tool for purification, selective enrichment as well as pre-concentration of biological samples prior to MS evaluation. SELDI is the technology using MALDI supports with different chromatographic affinities to specifically capture and enrich biomolecules as described for the ProteinChip Arrays (Ciphergen Biosystems, Fremont, CA, USA).³³ Compared with conventional MS-applications, the SELDI-technology is very easy to handle and timesaving regarding sample preparation and analysis.³⁴ In general this method consists of a SELDI Chip, a TOF mass analyser and software for data collection and analysis.³⁵ These chips offer a range of chemical affinities like hydrophobic, cation- and anion exchangers, IMAC, antibody-antigen and DNA-protein to immobilize specific proteins and to analyse and identify them through MS. Different mass fingerprints are generated from biofluids which can be furthermore classified as healthy or diseased by applying adequate bioinformatic tools.³⁶

Differences in protein-profiles of disease and control-related samples are caused by overexpression, abnormally shed proteins or protein fragments, modified proteins, proteolytically cleaved proteins or degradation due to the proteosome pathway.³⁷ There have been many examples of the use of SELDI for the determination of disease biomarkers, with the primary focus being diagnostics for many forms of cancer. During the last years SELDI technology achieved few authentic goals by claiming some potential biomarkers for Alzheimer disease,³⁸ amyotrophic lateral sclerosis (ALS) disease,³⁹ ovarian cancer⁴⁰ and prostate cancer.⁴¹ However, the enthusiasm for using this technology declined because of methodological and bioinformatic artifacts and biases found by other groups, which questioned the validity and reproducibility of the published results.^42,43

Additionally, concern has been raised about the long-term robustness of SELDI and the possible contributions of non-biological variation to the results.^44,45 A major question is: Is just mining out the differences between protein-patterns of healthy and diseased serums enough to incorporate this method into clinics? By performing solely protein profiling, predictions are only based on the comparative pattern analysis of healthy and diseased samples and the question still remains, whether these distinctive peaks, picked out through SELDI-MS without their identification, are clinically applicable for diagnostics. Another important consideration is that the specificity of biomarkers must be checked by comparing them from one disease with other similar diseases. Although SELDI technology is not easily applicable to identify candidate disease markers it can be used as a first step towards biomarker discovery for fast protein screening of biological relevant samples.