MALDI FT-ICR MS platform for proteomics: Rationale for an offline approach and optimised implementation

Posted: 19 July 2008 | Eric C. Peters et al, Genomics Institute of the Novartis Research Foundation | No comments yet

MALDI FT-ICR MS platform for proteomics: Rationale for an offline approach and optimised implementation
A number of sophisticated approaches have been developed to study the structure and function of genes, including the whole-scale sequencing of entire organisms[1], global transcriptional profiling[2], and forward genetic studies[3]. However, these techniques are ultimately limited by the fact that they only assess intermediates on the way to the protein products of genes that ultimately regulate biological processes[4,5].

MALDI FT-ICR MS platform for proteomics: Rationale for an offline approach and optimised implementation A number of sophisticated approaches have been developed to study the structure and function of genes, including the whole-scale sequencing of entire organisms[1], global transcriptional profiling[2], and forward genetic studies[3]. However, these techniques are ultimately limited by the fact that they only assess intermediates on the way to the protein products of genes that ultimately regulate biological processes[4,5].

MALDI FT-ICR MS platform for proteomics: Rationale for an offline approach and optimised implementation
A number of sophisticated approaches have been developed to study the structure and function of genes, including the whole-scale sequencing of entire organisms[1], global transcriptional profiling[2], and forward genetic studies[3]. However, these techniques are ultimately limited by the fact that they only assess intermediates on the way to the protein products of genes that ultimately regulate biological processes[4,5].

Processes such as RNA processing, proteolytic activation, and hundreds of possible post-translational modifications (PTMs) can result in the production of numerous proteins of unique structure and function from a limited number of genes. Additionally, biological activity often results from the assembly of numerous proteins into an active complex, the nature and composition of which can only be explored at the protein level. Therefore, proteomic studies should be able to answer many questions about cellular processes and diseases that cannot be answered by genomic methods alone6.

However, such studies are far harder to perform than their genomic counterparts, and any general analysis platform must possess high sensitivity, be tolerant of a wide range of experimental and analytical conditions, and be able to process and display massive amounts of information. More importantly, these analysis systems must be able to perform extremely high-throughput measurements, since unlike the relatively fixed nature of the genome, the expression and interactions of proteins are in a constant state of flux, varying over time, tissue type, and in response to environmental changes.

Historically, two-dimensional gel electrophoresis (2DE) has been the dominant technique for assessing large-scale changes in protein expression patterns7. The development8,9 and emergence of biological mass spectrometry (MS) in the early 1990s greatly increased the amount of information obtained using this technique, enabling the identification of thousands of encoded proteins by peptide mapping and/or tandem MS experiments10. Although powerful, the technique as practiced remains laborious, and possesses several widely recognised limitations, including the difficulty of comparing results between laboratories11, operational difficulty in handling certain classes of proteins, and potential unwanted chemical modifications12,13. More importantly, an even greater shortcoming of the classic 2DE technique is its inability to accommodate the extreme range of protein expression levels inherent in complex living organisms due to sample loading restrictions imposed by the gel-based separation technology employed14. This limitation is of particular concern in that most interesting classes of regulatory proteins are often expressed at low copy numbers per cell. Extensive protein prefractionation schemes based on differing solubility15,16, isoelectric point17, or subcellular location18 have been proposed to address the problem of analysing low abundance proteins. However, questions remain as to whether the integrity of the original protein mixture can be maintained, and all of these approaches greatly increase the number of relatively slow 2DE experiments that need to be performed.

These concerns have prompted a number of groups to explore alternative techniques to 2DE. For example, recognition of the fact that the full potential of MS methodologies is greatly under utilised in 2DE systems has led multiple groups to explore analysis platforms based solely on multi-dimensional chromatography combined with MS and/or tandem MS methods19,20. The chromatographic separations serve to disperse the complexity of the initial sample, and can be performed at both the peptide21 as well as at the protein level22, although protein identification is primarily performed using peptides23. Using these methodologies, the identification of encoded proteins present in affinity complexes, organelles, and even whole organisms has been performed24,25. Additionally, novel methodologies for reducing the complexity of the analyte mixture based on the presence of particular amino acids26,27,28 or specific PTMs27,29 have been employed to analyse even low abundance regulatory proteins or PTMs30,31,32. Similarly, techniques for assessing quantitative differential display of base proteins27,28 or PTMs30,33 between two samples have also been described.

A potentially powerful paradigm: LC MALDI MS

Although these liquid chromatography/MS-based approaches for the wide-scale analysis of protein expression patterns exhibit tremendous potential for improved analyses, they have to date overwhelmingly employed electrospray ionisation (ESI) methods due to the simplicity of their implementation. However, the operational parameters of ESI coupling methods also impose several limitations. Specifically, the separation system and mass spectrometer employed are coupled directly in real-time, making the construction of parallel analysis systems difficult (or at least extremely costly), and often preventing the mass spectrometer from continually collecting useful data due to the equilibration and washing periods typical of separation techniques. More importantly, current instrument control and data analysis software is not nearly fast enough to allow real-time data-dependent processing during the course of a chromatographic separation except when employing simple selection criteria such as peak intensity. This necessitates that upon the completion of a separation and subsequent analysis of the resulting data, the same sample must often be rerun to focus on those species that exhibited the desired selection criteria34. Additionally, targeted experiments monitoring the levels of several particular species over time still requires the active engagement of the mass spectrometer over the whole course of the chromatographic run, even though the species of interest themselves elute only in specific narrow time windows during the gradient profile. Ultimately, these and other limitations result in dramatic reductions in overall platform throughput.

In light of these considerations, several groups have begun to explore MALDI-based analysis platforms that involve the creation of a ‘permanent record’ of the multidimensional separation by depositing the effluents of the final separation columns directly onto MALDI target plates. Decoupling the separation step from the mass spectrometer in this manner guarantees that the chromatography can be performed free of artificially imposed restrictions, while the mass spectrometer can operate at maximum throughput. The resulting plates can also be reanalysed as required without the need to repeat the separation step, thus decreasing sample requirements while simultaneously greatly increasing the overall throughput of the system.

Clearly, the sample deposition method employed is critical to the success of creating high fidelity, reproducible ‘permanent records’ of the liquid chromatographic separations. ‘Heart fractionation’ methods35 are relatively simple to implement, but potentially sacrifice a significant part of the chromatographic resolution obtained during the separation. By contrast, sophisticated piezo-actuated microdispenser systems fabricated by the anisotropic wet etching of monocrystalline silicon have been described for the deposition of chromatographic effluents directly onto MALDI target plates36,37. Similarly, specialised liquid junction-coupled sub-atmospheric pressure deposition chambers for the off-line coupling of capillary electrophoresis (CE) with MALDI MS have also been described38,39. In light of the potential power of this offline approach, our group has developed a simple but robust fully automated system for the real time deposition of the effluents of mHPLC columns and matrix onto MALDI target plates.

An automated deposition system for LC MALDI MS approaches

Samples are first fractionated using such chromatographic modes as strong cation exchange, immobilised metal affinity, or amino acid specific enrichment methods based on the purpose of the overall experiment27. Regardless of the initial separation performed, the resulting column effluent is loaded directly onto a reversed-phase mHPLC column. The outlets of a series of these columns are held in parallel, and MALDI target plates clamped to an x,y translational stage are physically moved automatically underneath the columns during the course of the separation. It is important to note that the outlets of the columns and the target plates never come into direct physical contact. Instead, the effluents of the columns are transferred to the plates using an electrically mediated mechanism by applying a constant or intermittent negative potential to the target plates. This results in either a continuous stream or a series of droplets of precisely controlled volume respectively. In theory, the deposition of a continuous stream should more accurately maintain the fidelity of the separation process38,39. However, such an approach would also require the MALDI system to query the entire length of the trace, since eluting samples would not be localised to any particular position.

We use a 6144 Microtiter Format array deposition system. Roughly 150 nL aqueous droplets were precisely arrayed on a three by five square inch stainless steel plate in a 6144 microtiter array format, with each spot clearly distinguished from its nearest neighbours. Using this deposition system, matrix can also automatically be applied to the target plate before, during, or after the chromatographic process. Additionally, the deposition system works equally well with aqueous or numerous organic solvents, enabling the possibility of novel, automated on-plate recrystallisation processes no longer limited to solvent mixtures of acetonitrile and water that often further increase analyte sensitivity. This unique combination of automation and operational flexibility enables facile optimization of the many variables known to effect MALDI crystallisation processes.

In addition to the technical capabilities of the deposition system, our platform also makes use of specially patterned target plates consisting of hydrophilic regions arrayed on an otherwise hydrophobic surface40. After deposition of a sample onto a target region, both the analyte and matrix localise into an area far smaller than that occupied by the originally deposited droplet as the sample solvent evaporates, resulting in the effective concentration of the analyte on the hydrophilic regions. The use of such target plates provides considerable operational advantages in our analysis platform. For example, the sensitivities of ESI methods are known to be highly concentration dependent, often necessitating the use of nanochromatography to achieve maximum sensitivity19. Although effective, such nanoscale chromatography systems present numerous operational problems and often involve the manual loading of samples directly onto the separation column before it is connected to the HPLC system. By contrast, the surface-patterned target plates further concentrate the samples after the chromatographic process is complete, enabling the use of 300 mm id capillary columns and commercially available autosamplers.

In addition, the localisation of analytes to precisely defined locations approximately 400 mm in diameter on the target plates prevents the MALDI stage from querying areas that contain no analytes39. More importantly, increasing the size of the MALDI laser spot to approximately 400 micrometers allows the entire sample to be queried simultaneously, thus greatly reducing the problem of searching for ‘sweet spots’ often encountered when using the dried droplet method of sample preparation. Together, these factors greatly increase the sample throughput of the overall platform.

The unique capabilities of our deposition system have enabled the development of robust, optimised spotting methodologies for a variety of different matrices, including alpha-cyano-4-hydroxycinnamic acid (HCCA).

Novel chemical labels for increasing the MALDI ionisation efficiencies of peptides

Even after the issues involving the reproducible deposition of mHPLC column effluents with matrix onto target plates have been addressed, a MALDI-based analysis platform still presents several other distinct challenges. For example, the performance of MALDI mass spectrometry can greatly be affected by competitive ionisation effects, which are especially prevalent in complex mixtures41. The automated nature of our deposition system eliminates some of these problems by providing a reproducible environment for the recrystallisation of analyte and matrix molecules. In addition, MALDI has been shown to preferentially promote the ionisation of more hydrophobic peptides in complicated mixtures due to their enhanced co-crystallisation with the matrix42, thus providing an incomplete representation of a sample’s composition. However, since the peptide mixtures in our platform are subjected to reversed-phase mHPLC rather than CE immediately before deposition, all of the peptides deposited in an individual spot should exhibit nearly identical hydrophobicities, and therefore co-crystallise more similarly without resorting to complicated recrystallisation methods38,39. Nevertheless, there are other sources of ion suppression in MALDI-based methods that cannot be rectified solely by the deposition system.

Krause and co-workers investigated the dominance of arginine-containing peptides in the MALDI MS analysis of tryptic digests, and found that peptides containing an arginine residue exhibit a four- to eighteen-fold increase in signal intensity compared to those peptides containing a lysine43. This bias is thought to result from the higher basicity of the arginine residue compared to lysine. Greater sequence coverages could in theory be obtained using a labelling methodology that either increases the basicity of lysine residues44-48 or reduces the basicity of arginine residues49. However, the former method has attracted more attention, because reducing the basicity of arginine is considered detrimental to the overall sensitivity. Based on earlier work done with proteins50, several groups45-48 have recently reported the use of O-methylisourea to convert C-terminal lysine residues in tryptic digests to more basic homoarginine residues. In all cases, this treatment resulted in higher sequence coverages in peptide mapping experiments compared to the underivatised sample. Although effective in increasing ionisation efficiencies, this label is not amenable to the facile incorporation of inexpensive stable isotopic species for differential quantitation studies.

Therefore, our group developed a labelling reagent that increases the ionisation efficiencies of lysine-containing peptides while simultaneously enabling differential quantitation measurements51.

Peptide mapping experiments of tryptic protein digests after functionalisation with the above reagent showed total amino acid sequence coverages of nearly double those of their unlabeled counterparts51. It is important to note that although the term high-throughput has traditionally been equated with faster measurements, this labelling scheme also increases the overall throughput of the system, as it enables the collection of greater amounts of data in the same unit time.

In addition to its ability to affect increased MALDI ionisation efficiencies, this chemical label can further increase the efficiency of the analysis platform due to the isotopic substitutions that can easily be made at the two methylene ring carbons. The combination of a 4 Da mass difference per differentially labelled moiety in conjunction with the unique chemical selectivity of the label for lysine residues rather than the free a-amino group at a peptide’s N-terminus enables the user to accurately determine the number of lysine groups contained within a given peptide, and thus further limit the number of search possibilities when attempting to determine the identity of the protein from which it arose.


The sheer complexity and ever changing nature of the proteome necessitates the development of more powerful, higher-throughput analysis platforms. In theory, a MALDI-based LC/MS platform could favourably address many of the operational disadvantages inherent in the real time coupling of separation systems and mass spectrometers using ESI ionisation. However, in order to realise these potential advantages, numerous aspects of the MALDI process such as instrumentational issues, sample deposition, and chemical derivatisation need to thoroughly be investigated and optimised. The sample processing methods described above have been designed to maximise the throughput of any MALDI-based analytical platform, and are therefore compatible with a wide range of commercially available instrumentation.


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This article was originally published in American Pharmaceutical Review, Volume 5, Issue 3, on pages 72-81.