sample preparation of biological specimens is an essential part in any mass spectrometry- ( ms- ) based proteomics experiment since body fluids as well as tissue sections are highly complex in terms of variety in components and their concentrations .
a wide and diverse range of sample preparation strategies has been developed that provide sample ( protein ) fractions for further identification and quantification by ms .
the preferred route for sample prep obviously relates to the actual research question taking into account that any bias should be avoided . for comprehensive coverage of the proteome the isolation of as many peptides and/or proteins as possible is required .
however , for high - throughput ( htp ) purposes the capture of a small set of peptides can be sufficient in order to answer a specific question . in our laboratory ,
we have developed fully automated sample preparation pipelines that allow ms - profiling of body fluids at htp ( 5001000 samples per 24 hours ) and have been applied in clinical studies based on biobanked samples [ 24 ] . initially , most of these so - called peptide and protein profiles were acquired on matrix - assisted laser desorption ionization ( maldi ) time - of - flight ( tof ) mass spectrometers [ 5 , 6 ] .
although this combination still is extremely powerful in terms of costs and analysis time , we have recently shifted the ms - workflow to an ultrahigh resolution fourier transform ion cyclotron resonance ( fticr ) system in combination with maldi ( see figure 1 ) [ 7 , 8 ] .
the low part - per - million ( ppm ) mass accuracy of peptides and proteins observed between 1 and 9  kda in such fticr profiles facilitated sequence identifications while excellent case - control classification characteristics were conserved .
increasingly and in particular for case - control disease studies high - throughput analysis is required , that is , short measurement times or multiplexed analysis .
htp platforms that allow the analysis of large numbers of samples are needed in both a research setting and a hospital setting where doctors and patients demand fast answers .
efforts to optimize throughput parameters should therefore be considered during the design of a procedure .
furthermore , when performing quantification experiments it is important to reduce the number of handling steps prior to analysis , in order to obtain minimal loss of analyte , and to have a good estimation or , better , measurement of the efficiency of recovery .
blood samples are relatively easy to obtain and the concentration of protein / peptides in  blood - derived 
samples is high ( reference range for serum is 6085  gl total protein ) .
although urine can be obtained even in a noninvasive way , it is much less used in proteomics studies .
disadvantages of urine are the presence of ms - hampering salts and a lower concentration of proteins and peptides compared to plasma and serum , namely , normal total protein concentrations of about 20  mgl in a standardized sample based on healthy individuals .
moreover , proteins and peptides present in urine may not correlate with the state of the body and blood samples do , with the exception of , for example , kidney diseases [ 10 , 11 ] .
alternatively , cerebrospinal fluid ( csf ) is a relatively  clean  sample and can often be further used without extensive preparation steps .
it can however not be easily obtained and if so only in small volumes . in this paper various sample
previously , prefractionation techniques aiming for ( clinical ) ms - based proteomics have been overviewed extensively [ 1218 ] .
all workflows discussed in the current review are based on solid - phase extraction ( spe ) and can be combined with htp bottom - up as well as top - down proteomics approaches .
standardized body fluid sample collection and sample storage protocols are crucial for appropriate sample preparation .
the aim is to time - snap the state of the sample or , in other words , to keep the composition and quality of the sample as close as possible to the time point at which the sample was taken . in the case of serum and plasma , important parameters are processing times ( e.g. , the clotting time of a blood sample ) , storage times , storage temperatures , and number of freeze - thaw cycles [ 19 , 20 ] . for plasma samples additives such as heparin or citrate
it should be noted that the presence of heparin in a complex protein sample will influence chromatographic performance , as well as the anticoagulant ethylenediaminetetraacetic acid ( edta ) .
furthermore , it should be noted that clotting ( or coagulation ) factors are removed in serum samples ( listed at http://en.wikipedia.org/wiki/coagulation ) and that additional proteins or peptides with affinity to these clotting factors can be removed undesirably .
another variable that is of great importance in sample preparation involves the consumables that are used for collection and processing .
it is known that certain brands of plastic containers may release polymers or other interfering species .
for example , we have detected bovine serum albumin ( bsa ) from adhesive foils to close microtitration plates ( mtps ) as a major interfering compound .
proteins and peptides may also adhere to tube walls giving rise to significant differences in protein composition between two aliquots of the same sample processed in different tubes [ 22 , 23 ] .
solid - phase extraction ( spe ) is widely used in ms - based proteomics as a sample preparation technique . with spe compounds
are isolated on basis of their chemical and physical properties which determine their distribution between a mobile liquid phase and a solid stationary phase . after binding of the molecules with the correct properties ,
the remaining compounds are washed away and the bound molecules are eluted from the solid phase by changing the mobile phase into the elution solvent . whereas a chromatographic column for separating compounds by elution with a gradient is used multiple times , spe material is usually disposed after each sample and no gradient is applied for elution ( one - step elution ) .
thus in theory all compounds present in the sample are captured with chromatography , while with spe only a certain group of analytes is isolated , depending on the solid phase .
therefore spe is mainly used to clean up a sample and reduce sample complexity . for protein analysis with ms
it is often used to remove salts and other impurities that might cause ion suppression . however , by carefully choosing the right spe sorbent a higher selectivity can be achieved .
this selectivity also means that in principle with spe only a subfraction of the sample is analyzed , because not all compounds are captured , but only those compounds that match the binding capabilities of the sorbent .
spe material is available in various formats , including ( micro- ) columns , cartridges , plates , micropipette tips , and functionalized magnetic beads ( mbs ) .
first , their small size allows a higher concentration rate and therefore sensitivity due to their large binding surface area [ 27 , 28 ] .
second , the use of mbs can be automated , providing a highly consistent high - throughput method .
differences between tips become apparent when monitoring liquid handling performance in terms of back pressure upon wetting and elution of the tip . for this reason ,
the implementation of spe tips on a robotic pipetting system is not trivial ; that is , it is difficult to program a robot in such a way that each tip is sufficiently wetted and similar elution volumes are obtained .
for example , the default mixing feature in the software running our robotic system results in inadequate washing due to the slow liquid flow in the tip packed with spe material . to correct for this , a settling time is needed after the aspiration step as well as after the dispense step during the mixing cycle , similar to the procedure generally implemented for very viscous liquids .
examples of integrating spe cartridges in an ms - based bottom - up workflow have been reported [ 30 , 31 ] .
the most commonly used spe material in proteomics for the isolation ( or fractionation ) of proteins and peptides is reversed - phase and to a lesser extent ion - exchange material . for the isolation of glycosylated proteins and peptides normal
a summary of spe materials is given in table 1 , partially based on an earlier overview [ 24 , 34 ] .
less commonly used spe materials are for instance silica- or polystyrene - based [ 35 , 36 ] .
recent advances in ion - exchange chromatography have been made with mixed - bed materials .
mommen and coworkers combined weak anion - exchange ( wax ) with strong cation - exchange ( scx ) material . in this way
, they were able to perform a salt - free elution using a diluted formic acid solution to which dmso was added , while optimizing the wax / scx ratio .
peptide recoveries could be improved in comparison to scx alone with a wax / scx ratio of 4 .
this improved recovery could be explained by the so - called donnan effect , as well as by the absence of salts ( that are usually incompatible with ms ) by using a ph block - gradient .
this donnan effect also accounts for binding of proteins to newly developed core - shell nanoparticles .
core - shell nanoparticles are composed of a solid interface covered with a shell of charged polymers either as microgel or as densely grafted linear polymers ( so - called polyelectrolyte brushes ) . the exact mechanism that is involved in the binding of proteins to these nanoparticles
nanoparticles have successfully been applied in the isolation of low - abundance proteins , as not only are electrostatic interactions important , but these nanoparticles also have molecular sieving properties , which prevent large high - abundant proteins such as albumin from being captured
. further advantageous properties of these nanoparticles are their concentrating capacity and large surface area and their protectiveness against enzymatic degradation and other environmental influences on the captured peptides .
another specific example of solid - phase extraction of peptides and proteins involves the application of a solid - phase peptide library [ 42 , 43 ] .
such a library allows for the enrichment of low - abundant proteins , while simultaneously reducing the relative concentration of abundant species , without needing protein depletion . in this approach a complex protein sample
is exposed to a ligand library in large overloading conditions , resulting in a rapid saturation of each bead with affinity to an abundant protein and a remaining vast majority of the same protein in solution ( i.e. , unbound ) [ 44 , 45 ] .
the beneficial aspect is that trace proteins will not saturate the corresponding partner beads but are captured in progressively increasing amounts .
this process of equalization of the large range in protein concentrations or compression of the sample dynamic range is commercialized with the proteominer protein enrichment kit .
this combinatorial hexapeptide library has been applied in various ms - based proteomics studies ( including at our own laboratory ) and it was found that the large variation in protein concentrations was decreased in complex biological samples , thus resulting in a relative enrichment of medium- and/or low - abundant proteins [ 4655 ] .
generally , the increment in the number of species detected was from twofold to fivefold compared with control , nontreated samples .
this substantial increment in detection applies mostly to low - abundance proteins , considering that they could not be detected in the untreated samples , where they have been obviously present . in our laboratory , we have implemented various spe protocols ( based on mbs ) in a modular way on a liquid handling hamilton robot . in addition , for bottom - up proteomics purposes , a protein digestion protocol has been implemented on the same robotic platform allowing both in - gel or in - solution digestion . in the first case ,
the digestion protocol starts with several washing steps of the gel - plugs , followed by reduction / alkylation of the proteins and peptides and additional washing steps of the gel - plug , followed by incubation with a proteolytic enzyme at elevated temperatures and an acidification step to deactivate the enzyme . in the case of in - solution digestion
the digestion protocol can be continued with a peptide purification step by means of spe , generally in the form of a reversed - phase c18-tip .
on our robotic system , purification with c18-mbs has proven to be a powerful method .
moreover , cartridge - based spe protocols for protein extraction were developed and automated on a spark symbiosis system .
affinity enrichment is based on the reversible and noncovalent interaction of the protein or peptide of interest with naturally occurring ( or synthetic analogues of ) biomolecules , such as other proteins , lectins , nucleotides , or dyes .
affinity enrichment is a popular chromatographic method for the immunocapture of compounds in larger quantities , for instance , immunoglobulins ( iggs ) with prota or protg .
it is however also a convenient method for spe of smaller sample volumes aiming for capture of a specific protein or peptide ( see figure 2 ) . as presented in the previous section and in table 1 , various materials for affinity enrichment are available .
moreover , immunocapture strategies can also be used for depletion of high - abundant proteins to allow access to the low - abundant ones ( in - depth analysis ) . for this purpose ,
commercially available products make use of a mix of antibody - based affinity material , such as the proteoprep 20 plasma immunodepletion kit from sigma , which removes the 20 most abundant proteins in a single purification step , or mars from agilent .
qian and coworkers used the commercially available proteomelab igy12 affinity column ( beckman coulter , fullerton , ca ) in combination with a custom made supermix column to separate more than 50 high- and medium - abundant proteins from the lower - abundant ones in a plasma sample . in the case of enrichment strategies , in general more information about the analyte of investigation is already available and the research question has focused on a very specific group of proteins or even a single protein .
an elegant example of immunoaffinity ( ia ) enrichment was shown by halfinger and coworkers , which involved a thermal protein precipitation step , followed by liquid - liquid precipitation before ia chromatography to quantify the low - abundance heart failure marker n - terminal pro - b - type natriuretic peptide ( nt - probnp ) .
this example showed a combined preparation method to deplete high - abundant proteins followed by concentration of a specific low - abundant protein through immunoaffinity .
the simple and cost - effective method could be used to unmask low - abundant proteins in general .
finally , it should be noted that another important and widely used application of affinity purification concerns the isolation of phosphoproteins through metal - affinity spe and that the isolation of glycoproteins or glycopeptides through lectin - affinity is a field of their own .
a combination of protein digestion and affinity enrichment spe of a peptide is used in the so - called workflow of siscapa , which stands for stable isotope standards with capture by anti - peptide antibodies .
this targeted protein quantification approach was coined in 2004 by anderson and coworkers   and comprises the isolation of a known ( proteolytic ) peptide ( from a clinically relevant protein ) by means of antibody coated spe material after proteolysis of the total sample proteome .
where protein digestion was originally used in the discovery stage of ms - based proteomics studies to allow protein identifications , siscapa aims for absolute quantification of the analyte in a clinical setting .
such quantification relies on the use of external calibrators , such as a stable - isotope - labelled peptide ( with identical amino acid sequence ) , in order to enable determination of the concentration of the released proteolytic peptide and thus the endogenous protein . for this purpose ,
precise and detailed knowledge on the extent and reproducibility of the digestion is crucial . with regard to sensitivity and
limit - of - detection , a number of ia techniques in combination with ms have been reviewed .
although elisa still outperforms ms - based techniques in terms of sensitivity , the potential of siscapa to allow quantification of low - abundant proteins in body fluids is generally acknowledged .
recently , the siscapa workflow has been automated by means of magnetic beads and concurrently improved sensitivity to the pgml range by increasing the sample volumes [ 31 , 66 , 67 ] .
similar methodologies have been proposed , based on immunocapture at the protein level . here , the wider availability of antibodies against many proteins could be advantageous .
it is further suggested that sensitivity improves with the number of so - called proofreading steps .
a proofreading step is regarded as a step in an assay that results in a stepwise decrease in the error fraction .
we foresee that the combination of the two techniques mentioned by ackermann and berna will add extra proofreading steps ( two different antibodies instead of one ) and together with the improvements in siscapa workflows will increase sensitivity even more .
high - throughput analysis is required in particular for clinical samples when ms - based proteomics is applied to case / control disease studies .
sample preparation strategies are aimed at reducing sample complexity and improving quality of the measurements .
the automation of protein purification methods has become crucial in order to balance the speed of ms - acquisition for both bottom - up and top - down proteomics analyses .
processes on a robotic platform should be implemented in a modular way to allow flexibility in choosing spe materials and solvents .
the selection of an spe method should be based on the potential for automation . for exploratory as well as for screening studies , general solid phase materials such as rpc18 and scx
are widely used , whereas a targeted approach requires a specialized solid phase such as affinity spe material .
high - throughput strategies have been developed for spe materials on various formats , including cartridges , micropipette tips , and functionalized magnetic beads ( mbs ) .
 for large - scale and standardized applications in mass spectrometry- ( ms- ) based proteomics automation of each step is essential . 
 here we present high - throughput sample preparation solutions for balancing the speed of current ms - acquisitions and the time needed for analytical workup of body fluids . 
 the discussed workflows reduce body fluid sample complexity and apply for both bottom - up proteomics experiments and top - down protein characterization approaches . 
 various sample preparation methods that involve solid - phase extraction ( spe ) including affinity enrichment strategies have been automated . obtained 
 peptide and protein fractions can be mass analyzed by direct infusion into an electrospray ionization ( esi ) source or by means of matrix - assisted laser desorption ionization ( maldi ) without further need of time - consuming liquid chromatography ( lc ) separations . 
1. Introduction
2. Body Fluid Sample Collection
3. Solid-Phase Extraction of Proteins and Peptides
4. Affinity Enrichment of Proteins and Peptides
5. Conclusions