Analysis of biopolymer structure and post-translational modifications by mass

Unveiling structure – function relationships of biologically relevant molecules has been a main theme of analytical biopolymer chemistry for decades, and an ultimate goal of all recent analytical developments is to contribute to the understanding of life. The classic techniques of structure determination, X-ray crystallography and NMR, have established important milestones in life sciences. Some outstanding examples of results in X-ray crystallography are noteworthy, such as the structural elucidation of the ribosome, nucleasome, photosystem I, GroEL – GroES, a bacterial potassium ion channel [117-121].

NMR has revealed information about dynamic processes that occur in solution, for example when a ligand binds to a protein; these results owe to the Nobel Prize-awarded observation that the Nuclear Overhauser Effect can be exploited to map networks of near-by atom pairs that are not connected through covalent bonds [122-124].

Over the past two decades, mass spectrometry (MS) has become an essential tool in structural biology, due to its ability to provide molecular structure information and to complement other analytical methods. Milestones in MS have been recently achieved with the introduction of soft-ionization techniques, electrospray (ESI) [125, 126] and matrix assisted laser desorption ionization (MALDI) [127], that enabled analysis of large biopolymers in gas-phase with high sensitivity and accuracy. Factors such as high sensitivity, low sample consumption, low analysis time and applicability to mixtures make MS a method of choice for many analytical problems in life sciences, where conventional methods reach their limits. In the electrospray process (reviewed in reference [126]) multiply charged analyte molecules are produced by spraying a solution containing the molecules of interest through a thin needle that has a potential difference applied to it, with respect to the counter electrode. The analyte can be an intact protein, small peptide, DNA, lipid or carbohydrate. As very low residual energy is retained on the analyte upon ionization, ESI represents a soft ionization technique capable of preserving the tertiary structure of proteins in the gas phase. This enables the analysis of intact protein complexes [128-130] and, most recently, whole virus assemblies [131-133]. Moreover, in the absence or complementary of NMR or X-ray data, information about surface topology, folding pathways of proteins and protein – protein interactions can be derived from mass spectrometric data. Several approaches have been developed, including differential chemical modification of specific amino acid residues in proteins [134-138] and oxidative

protein footprinting [139-143]. These are based on the assumption that the rate of modification of a specific amino acid is dependent on the inherent reactivity of a side chain and on its solvent accessibility, i.e. surface exposed residues react at a higher rate than residues berried in a hydrophobic pocket. In addition, the use of bifunctional cross-linking reagents can provide information regarding distances between residues in protein [144-146], reviewed in [147].

1.7.1 Mass spectrometric approaches for structural characterization of antibodies Affinity – mass spectrometry methods, initially developed by our laboratory [136, 148, 149] have now been successfully established by many research groups for the identification of molecular recognition structures in proteins and other biopolymers, such as antigenic determinants – epitopes – recognized by antibodies paratope regions [150-152] (reviewed in [153]). The method, referred to as proteolytic epitope excision, involves the covalent immobilization of a biopolymer, e.g. an antibody or a ligand on a stationary phase. The antigen or a mixture containing the antigen is presented to the column, resulting in an affinity-bound antibody – antigen complex. The basis for this approach is that the molecular recognition structures involved in the antigen – antibody interactions – the epitope and the paratope – are shielded against chemical reagents, such as proteolytic enzymes. Differential proteolytic digestion of the immuno-complex will result in antibody bound peptide epitope(s) that can be subsequently dissociated and identified by MS (see Figure 1.10). In the Aspecific antibodies, this methodology was applied to identify the ß-amyloid epitopes recognized by Aß-antibodies with distinct serological and therapeutic properties [79, 91]. The results explained the major differences between plaque-resolving and plaque-protective antibodies, whereas follow-up studies pursued the development of immunotherapeutic agents containing the epitope lead structures defined by MS [154, 155].

Figure 1.10: Principle of mass spectrometric epitope identification by proteolytic excision of epitopes in immune complexes: the antigen-antibody complex is subjected to enzymatic degradation, leaving the antigenic determinant affinity-bound to the antibody complementary determining regions. Following the dissociation step of the complex, MS analysis provides the molecular mass information of the epitope.

Most recently, affinity-MS has been employed for the first time in the reversed fashion to identify the paratope of the camel anti-lysozyme antibody cAbLys3 [156]; camel antibodies lack the light chains and the heavy chain CDR3 is considerably larger than the corresponding region in human or mouse immunoglobulins [157]. In this experimental setup an intact antibody (paratope excision) or its enzymatic mixture (paratope extraction) is presented to an affinity column containing the covalently immobilized epitope peptide.

Several challenges are associated with this approach, such as (i) the proteolytic stability of immunoglobulins, which may require their partial denaturation for an efficient enzymatic degradation, (ii) the simultaneous involvement of all six antibody CDRs in binding, that may result in loss of affinity of the peptides containing individual regions derived from enzymatic procedures during paratope extraction; and (iii) the lack of knowledge of most antibody sequences, derived in part from the complexity of the molecular mechanisms generating antibody diversity. Furthermore, mutations, truncations and post-translational modifications may complicate the analysis.

An alternative of the paratope excision/extraction approach is the primary structure determination of antibodies by mass spectrometry. Several MS-based approaches,

including so called “top-down” and “bottom-up” [158-160], are now routinely applied in the pharmaceutical industry for quality control purposes [161] (see Figure 1.11).

Figure 1.11: Principles of “top-down” (left) and “bottom-up” analysis (right) for structural characterization of antibodies.

Top-down MS refers to an approach where the intact molecule is introduced into the mass spectrometer without enzymatic or chemical fragmentations in-solution, and structural information is obtained from the fragmentation pattern of the intact molecule inside the mass spectrometer. Structural information can be obtained for variable and terminal regions of the antibody; however, its size represents a major challenge for this method. In the bottom-up approach, the protein is digested into small peptides by a protease, followed by liquid chromatography – tandem MS analysis (LC-MS/MS). The antibody sequence is assembled based on the molecular mass and sequence information of individual peptides and predicted amino acid sequence. A representative example of recombinant immunoglobulin is rituximab, a chimeric anti-CD20 mouse/human IgG1 monoclonal antibody produced in CHO cells – the first therapeutic antibody approved for the treatment of non-Hodgkin’s lymphoma. When the antibody's cDNA information is unknown, as is the case of immunoglobulins produced by hybridoma technology, primary structure determination may become a challenging task. Recently, we showed that de novo

interpretation of tandem MS data in combination with N-terminal Edman sequencing of proteolytic antibody peptides can be successfully employed for primary structure determination of antibodies of unknown sequence [162].

Following protein biosynthesis in the ribosomes, a large diversity of post translational modifications (PTMs) may occur expending the proteome diversity. These events finely tune proteins to effect specific biological functions. They usually occur at sub-stoechiometric levels and may be highly heterogeneous. Certain PTMs are permanent, such as N-glycosylation of asparagine residues, whereas others are transient, such as acetylation and methylation, phosphorylation or O-linked N-acetyl glucosamine, serving in the cellular signalling pathways. Knowledge of sites and nature of such modifications in proteins represent critical information for understanding of protein structure and function.

Whereas unmodified proteins can be studied by X-ray crystallography and NMR, these methods often fail to provide satisfactory information about post-translational modified proteins. For example, attachment of a carbohydrate moiety on a protein may lead to increased flexibility of the backbone around the attachment site, such that glycans often need to be trimmed to obtain decent diffracting crystals [163].

In the last two decades, tandem mass spectrometry using collision induced dissociation (CID) and electron capture/transfer dissociation (ECD/ETD) [164-167], described in detail in the following chapters, has emerged as a powerful tool for identification and molecular characterization of PTMs, providing information about their occupancy and site-specific microheterogeneity. Hyphenated methods combining separation techniques and (tandem) mass spectrometry are particularly suitable for analysis of complex mixtures containing modified and non-modified peptides, as they offer an additional “degree of freedom” to the analytical measurement. In addition, the dynamic range, i.e. the ratio of highest to lowest sample concentration that can be detected under identical analytical conditions, has been significantly extended.