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Two-dimensional electrophoresis (2-DE) is a high-resolution technique for analysis and comparison of complex protein mixtures. With the advent of recent technical developments, its application has become significant in a wide range of fields. This chapter describes a proteomic approach for the analysis of metastasis-associated proteins using pre-fractionation of glycosylated proteins via lectin (HPA) affinity chromatography prior to separation by 2-DE. Guidelines for the preparation and storage of buffers, experimental conditions and protocols of affinity chromatography, isoelectric focussing, and SDS-PAGE conditions are provided. Critical parameters associated with the different steps of 2-DE are discussed.

Most embryo culture media are still supplemented with proteins rather than with nonprotein macromolecules or recombinant protein products. HSA is probably the most common supplement followed by globulin-enriched preparations. Serum supplementation and Co-Culture of embryos belong to the past. Defined nonprotein or recombinant protein supplements are becoming a viable alternative during gamete and embryo manipulation procedures. Biological protein supplements are still preferred for any extended period of embryo culture. Understanding the goals and purpose of supplemented macromolecules in embryo culture media during each step of the laboratory IVF process should assist us in choosing the safest and most consistent macromolecule for each step, but also selecting a product that has the capability of delivering the best clinical outcome. Each batch of biological protein supplement is unique, even if supplied by the same manufacturer. Each lot of protein supplement typically contains many lot-specific, potentially harmful, and unintended hormone and protein contaminants. Macromolecular embryo culture medium supplements should be identified as one of the highest risk factors in an IVF laboratory that may contribute towards clinical compromise. All efforts should be made to use a proven batch of supplement for as long as the expiration date will allow. The beneficial effect of more complex protein supplements is evident after the activation of the embryonic genome and probably due to the presence of growth factors. Lower live-birth rates due to suboptimum protein supplementation may be a direct result of the preferential loss of female embryos. When deciding on a culture system, thought should be given specifically to the interaction between the culture medium and the macromolecular supplement. Ready-to-use pre-supplemented culture media may be advisable over a more complex product if a comprehensive macromolecular quality management program is not feasible. However, the question remains as to whether the increasing simplification of embryo culture media supplements is ready for large-scale clinical use.

Two-dimensional electrophoresis (2 DE) is one of the most important proteomic tools and allows studying the complexity of proteomes of different origin. This chapter describes a setup for 2D DIGE with minimal labeling for qualitative and quantitative applications. It relies on homemade gels of medium size and in our hands has been found useful for a wide variety of separation problems involving complex protein mixtures of animal or human origin. The basic method is given for serum proteins of different species, but with minor modifications the method may be easily adapted to other sample materials (other body fluids, cells, tissues), conditions, or size. Examples are given for simple pattern comparisons (e.g., quality control, fast comparison of just two samples) as well as for quantitative applications to larger sample sets.

Benzophenone photoprobes represent powerful tools for chemical proteomics. Upon UV irradiation, a benzophenone photoprobe can selectively form a covalent bond with its target protein in complex protein mixtures. Thus, photoprobes can be used to profile a wide variety of proteins in complex proteomes. This chapter describes simple protocols to derivatize fluorenylmethyloxycarbonyl (Fmoc)-protected peptide-nucleic-acid adenine (PNA adenine) into a benzophenone photoprobe and its application in photolabeling its target proteins. The method as described does not require specialized equipment for probe synthesis and photolabeling. In addition, the strategy is applicable to recognition motifs other than PNA adenine, such as peptides, to profile their target proteins in complex proteomes.

To sensitively analyze complex protein mixtures by mass spectrometry-based shotgun proteomics, researchers have employed platforms that couple orthogonal peptide fractionation methods using nanoscale HPLC. Commonly used platforms have coupled either strong cation exchange (SCX) HPLC or preparative isoelectric focusing (IEF) with nanoscale reversed-phase (nanoRP) HPLC fractionation of peptides. Coupling two dimensions of peptide fractionation, prior to mass spectrometric analysis, increases the sensitivity for identifying low abundance proteins. However, the large dynamic range of protein abundance and high level of complexity of protein mixtures derived from many biological sources, such as bodily fluids, require additional steps of peptide fractionation. To address this shortcoming, we have developed a platform combining three dimensions of peptide fractionation as follows: (1) preparative IEF; (2) SCX HPLC; and (3) nanoRP HPLC. This platform significantly increases the sensitivity of shotgun proteomic analysis in complex protein mixtures. Here, we describe the implementation of this three-dimensional peptide fractionation platform for proteomic studies of complex mixtures.

Selected reaction monitoring (SRM) is one of the most powerful techniques for the relative and absolute quantification of proteins from complex protein mixtures. In contrast to traditional protein quantification methods such as ELISAs or RIAs, the SRM method uses mass spectrometry for detection. Further benefits of SRM are as follows: (1) high specificity and sensitivity; (2) large linear dynamic range of at least three orders of magnitude; and (3) the possibility to quantify multiple proteins simultaneously in a single MS run from an individual sample. To perform SRM-based protein quantification reliably, a careful design of the assay is essential, and several pitfalls must be avoided. The aim of this chapter is to help SRM newcomers to establish SRM-based protein quantification assays and discuss an overview of typical work flows that are applied during SRM assay development.

The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into a mass spectrometer for protein identification and quantification. An integrated protein separation platform, combining capillary isoelectric focusing (CIEF) with reversed phase liquid chromatography (RPLC), is described to provide high resolving power for the analysis of complex protein mixtures. Thus, the proteins are systematically resolved according to their differences in isoelectric point and hydrophobicity using combined CIEF/RPLC separations. A key feature of the CIEF-based multidimensional separation platform is the elimination of protein loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein profiles within small cell populations or limited tissue samples.

LC-MS/MS is one of the most powerful techniques in the field of proteomics allowing high throughput identification of proteins out of complex protein mixtures. Besides high sample throughput, the analytical sensitivity is one of the major benefits of this technology. A prerequisite for sensitive LC-MS/MS approaches is chromatography with very low flow rates in the nanoliter per minute range, usually referred to as nano-liquid chromatography (nano-LC). However, to perform this separation technology, an appropriate instrumental setup as well experienced operators are a prerequisite. The aim of this chapter is to help nano-LC newcomers to get introduced to this fascinating technology. Technical components of nano-LC systems like solvent delivery systems, sample injection systems, and nano-chromatography columns are described. Detailed procedures to mount, test, and operate the system are outlined, and advices for an effective troubleshooting are provided.

The detailed characterization of complex protein mixtures as in samples from biological sources cannot be sufficiently performed by separation of polypeptides according to their molecular weight as is done by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1DE). For analysis of such samples, 2-dimensional gel electrophoresis (2DE) is the preferable methodological approach because it combines separation of polypeptides according to isoelectric properties and molecular weight as well. The resulting pattern of protein spots does not only provide information on composition of samples because of the complexity of a mixture of polypeptides. It delivers also a picture on the microheterogenity of polypeptides caused by post-translational modifications. These might be of natural or artificial type and occur during biosynthetic processing of a polypeptide or within industrial scale production. The presented method describes an experimental approach to investigate the influence of glycosylation in general and sialylation exclusively on spot pattern of proteins separated by 2DE.

Because the dynamic range of most cell or tissue proteomes is enormous, separation of such complex protein samples by two-dimensional electrophoresis (2-DE) on broad pH gradients often results in the visualization of only the most abundantly expressed proteins. It is, therefore, often beneficial to first subdivide the proteome in smaller, less complex fractions before 2-DE. This enables the analysis of a larger number of proteins. One approach to prefractionate protein samples is by reversed-phase high-performance liquid chromatography (RP-HPLC), separating proteins according to their hydrophobicity. This effectively introduces a third separation dimension, increasing the spatial resolution of the experiment. Here, we will describe a procedure for separating whole protein lysates by RP-HPLC, before their analysis by 2-DE or 2-D difference gel electrophoresis.