Human body fluids have been rediscovered in the post-genomic era as great sources of biological markers and perhaps particularly as sources of potential protein biomarkers of disease. Analytical tools that allow rapid screening, low sample consumption, and accurate protein identification are of great importance in studies of complex biological samples and clinical diagnosis. Mass spectrometry is today one of the most important analytical tools with applications in a wide variety of fields. One of the fastest growing applications is in proteomics, or the study of protein expression in an organism. Mass spectrometry has been used to find post-translational modifications and to identify key functions of proteins in the human body. In this study, we review the use of human body fluids as sources for clinical markers and present new data that show the ability of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to identify and characterize proteins in four human body fluids: plasma, cerebrospinal fluid (CSF), saliva, and urine. The body fluids were tryptically digested without any prior separation, purification, or selection, and the digest was introduced into a 9.4 T FTICR mass spectrometer by direct-infusion electrospray ionization (ESI). Even though these samples represent complex biological mixtures, the described method provides information that is comparable with traditional 2D-PAGE data. The sample consumption is extremely low, a few microliters, and the analysis time is only a few minutes. It is, however, evident that the separation of proteins and/or peptides must be included in the methodology, in order to detect low-abundance proteins and other proteins of biological relevance.

A procedure for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) of proteins is presented. The use of a newly presented capillary coating, PolyE-323, provided fast separations of typically a few minutes with high efficiency, good deactivation, and no bleeding into the mass spectrometer. Capillaries coated with PolyE-323 showed high stability over a range of pH 2-10, and tolerance towards methanol and acetonitrile, two modifiers commonly used in CE-ESI-MS. Due to the speed and simplicity of the coating procedure, the polymeric surface could, if necessary, easily be regenerated. This capability is especially valuable when working with samples of complex matrix, where a capillary surface cleaning step might be desired in order to eliminate possible memory effects. The potential of PolyE-323-coated capillaries in bioanalysis using CE-ESI-MS was demonstrated by analyzing peptides and proteins up to 66 kDa using time of flight (TOF)-MS. Due to the stable, anodal electroosmotic flow generated by the coating, the use of a sheathless ESI interface was enabled, demonstrated in peptide analysis with attomole sensitivity. The fast on-line CE-ESI-TOF system using PolyE-323-coated capillaries provided efficient separation and detection of a large number of peaks in a short time, exemplified by the analysis of a tryptic digest of bovine serum albumin (BSA). The capability of the developed capillary surface coating was demonstrated by the separation of human plasma and cerebrospinal fluid (CSF).

We have compared the brain proteome in the temporal neocortex between Alzheimer's disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.

A graphite-polyimide mixture was used as a conductive coating for sheathless electrospray emitters. The coating procedure described is simple and inexpensive compared to previously described methods. An investigation of the stability of the conductive coating carried out by electrochemical methods revealed good performances during oxidative stress. In addition, no decrease in emitter performance was seen during continuous electrospray in the positive electrospray mode for two weeks. Fast capillary electrophoresis with attomole sensitivity demonstrated the excellent performance of the described sheathless interface when used in conjunction with an orthogonal time-of-flight mass spectrometer. The overall simplicity, stability and low cost of this type of sheathless emitter make the described approach highly suitable for any on-column coupling of low flow rate separation techniques to a mass spectrometer.

There is growing interest in sampling of protein biomarkers from the interstitial compartment of the brain and other organs using high molecular cutoff membrane microdialysis (MD) catheters. However, recent data suggest that protein sampling across such MD membranes is a highly complex process that needs to be further studied. Here, we report three major improvements for microdialysis sampling of proteins in complex biological matrixes. The improvements in this in vitro study using human ventricular cerebrospinal fluid as the sample matrix include increased fluid recovery control, decreased protein adsorption on the microdialysis membrane and materials, and novel quantitative mass spectrometry analysis. Dextrans in different concentrations and sizes were added to the perfusion fluid. It was found that dextrans with molecular mass 250 and 500 kDa provided a fluid recovery close to 100%. An improved fluid recovery precision could be obtained by self-assembly triblock polymer surface modification of the MD catheters. The modified catheters also delivered a significantly increased extraction efficiency for some of the investigated proteins. The final improvement was to analyze the dialysates with isobaric tagged (iTRAQ) proteomics, followed by tandem mass spectrometric analysis. By using this technique, 48 proteins could be quantified and analyzed with respect to their extraction efficiencies. The novel aspects of microdialysis protein sampling, detection, and quantification in biological fluids presented in this study should be considered as a first step toward better understanding and handling of the challenges associated with microdialysis sampling of proteins. The next step is to optimize the developed methodology in vivo.

Hybrid capillary-poly(dimethysiloxane)(PDMS) microchips with integrated electrospray ionization (ESI) tips were directly fabricated by casting PDMS in a mould. The shapes of the emitter tips were drilled into the mould, which produced highly reproducible three-dimensional tips. Due to the fabrication method of the microfluidic devices, no sealing was necessary and it was possible to produce a perfect channel modified by PolyE-323, an aliphatic polyamine coating agent. A variety of different coating procedures were also evaluated for the outside of the emitter tip. Dusting graphite on a thin unpolymerised PDMS layer followed by polymerisation was proven to be the most suitable procedure. The emitter tips showed excellent electrochemical properties and durabilities. The coating of the emitter was eventually passivated, but not lost, and could be regenerated by electrochemical means. The excellent electrochemical stability was further confirmed in long term electrospray experiments, in which the emitter sprayed continuously for more than 180 h. The PolyE-323 was found suitable for systems that integrate rigid fused silica and soft PDMS technology, since it simply could be applied successfully to both materials. The spray stability was confirmed from the recording of a total ion chromatogram in which the electrospray current exhibited a relative standard deviation of 3.9% for a 30 min run. CE-ESI-MS separations of peptides were carried out within 2 min using the hybrid PDMS chip resulting in similar efficiencies as for fused silica capillaries of the same length and thus with no measurable band broadening effects, originating from the PDMS emitter.

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