Protein quantification using mass spectrometry

In discovery experiments large numbers of proteins are identified with no relevance to the biological question posed, whilst some proteins which may be relevant can be missed. Furthermore the absence of a protein from the identification list does not mean the protein is absent from the sample. It has been suggested that if reproducible and quantitatively accurate data can be generated for all proteins that are involved in a particular process then proteomics will make a greater impact in

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biology (Ren et al., 2006). Thus a quantitative approach to proteomics identifying and quantifying sets of defined proteins will progress our understanding of biological systems and disease states.

There are three principle methods used to compare amounts of proteins using MS. These are relative quantification, absolute quantification and protein kinetics or turnover. In addition two approaches can be used for quantitative; the utilisation of stable isotope labelling and’ label-free’ techniques.

a. Relative Quantification

Relative quantification measures changes in expression levels. Numerous quantitative methodologies have been dedicated to the extensive comparison of multiple proteomes commonly using isotopic-labelling approaches including stable isotope labelling with amino acids in cell culture; SILAC (Aebersold and Mann, 2003), isotope-coded affinity tagging; ICAT (Shiio and Aebersold, 2006). These methods involve labelling peptides, with reagents or labels that are chemically identical but vary in their isotope composition, prior to the sample being run on LC- MS. In addition isobaric tagging reagents have been used known as iTRAQ (isobaric tagging for relative and absolute quantification) (Wiese et al., 2007)and ‘label-free protocols’ have also been implemented (Wong and Cagney, 2010).

Label-free techniques have some advantages over labelling approaches. Although they require high mass precision instruments they do not require expensive isotope labels, or particular software and they can analyse many proteins in a single sample and many samples in a single experiment concurrently. Label-free quantitation uses either area under the curve, relying on signal intensity founded on precursor ion spectra or spectral counting which uses the number of peptides assigned to a protein in an MS/MS study (Zhu et al., 2010). The spectral counting approach uses data processing involving applications such as ‘exponentially modified protein abundance index’ or emPAI (Ishihama et al., 2005).

42 b. Absolute Quantification

Systems biology approaches, which require the ability to make quantitative measurements have furthered the demand for absolute protein abundance values for input into models (Otto et al., 2012). Absolute quantification is a ‘targeted approach’ as it focuses on a specific set of peptides. Thus prior information is required in order to produce MS assays for the detection and quantification of predetermined analytes in a mixture. As the method is hypothesis driven, a subset of peptides uniquely associated with the proteins of interest are targeted. In absolute quantification copies per cell of a protein from a cell lysate or molar quantities of a protein from a secretome or media can be determined. One advantage of this method is that it allows the real comparison of data between laboratories. Such precise determination of concentrations requires internal standards which must perform in the linear range of the system. The three types of isotope-labelled quantification standards are AQUA (for absolute quantification) (Gerber et al., 2003), QconCAT (quantification concatamers) (Beynon et al., 2005) and PSAQ (protein standard absolute quantification) (Dupuis et al., 2008) standards. In the latter a full-length protein with homologous biochemical properties of the target protein are spiked in at the start of the analytical process.

i. AQUA

In 2003 a strategy known as Protein-Aqua was presented for absolute quantification by using isotopically labelled peptides for downstream analysis by LC- MS (Gerber et al., 2003). This method, also known as stable isotope dilution (SID) is regarded as the gold standard for quantitation by LC-MRM-MS (Pan et al., 2009). Commonly isotope-labelled internal standards are used to measure small molecules such as hormones in analytical chemistry. The method employs de novo chemically synthesised, isotopically labelled internal standards. Interestingly due to the chemical synthesis of the peptide standards, AQUA has enabled quantification of post-translational modifications and biomarkers (Cummings et al., 2008). Peptides are quantified by monitoring either accurate mass retention time (AMRT) (Silva et

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al., 2005) or monitoring multiple reaction monitoring (MRM) transitions for each peptide (Figure 1.12).

i. QconCAT

A method using artificial concatamers of a set of standard peptides (Q-peptides) was introduced by Beynon et al. in 2005 (Beynon et al., 2005); QconCAT. This extends the number of proteins that can be quantified in parallel. A synthetic gene is produced which is then inserted into plasmid DNA. At least two peptides per protein are incorporated into the QconCAT in order to reduce the risk associated with poorly ionizing or difficult peptides (Pratt et al., 2006).

Figure 1.12. Mass spectrometry methodology using AQUA and multiple reaction monitoring (MRM). MS is undertaken using a triple quadrupole. At a set elution time the first quadrupole (Q1) is used to target and detect the parent ion of a given peptide (both heavy; AQUA peptide and light; native peptide co-elute at the same time from the LC column). This is fragmented by the collision cell, product ions are detected by the third quadrupole (Q3).

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In addition to the Q-peptides, the artificial protein contains a C-terminal

hexahistidine-tag (His-Tag) sequence for purification, fibrinopeptide following the initiator methionine and glufibrinopeptide ‘EGVNDNEEGFFSAR’ for quantification of the QconCAT. The fibrinopeptide and glufibrinopeptide also allow assessment of expression of the QconCAT as if both are present in a QconCAT digest this means the full length protein has been expressed. The genes are subsequently expressed in Escherichia coli (E.Coli) grown in media containing the selected label. Where trypsin is the proteolytic enzyme of choice for digestion L-lysine (13C6, 15N2) and L-

arginine (13C6, 15N4) are the labels of choice providing a 6Da mass shift between

standard and analyte. Following proteolysis each Q peptide is released in stoichimetry of 1:1. Subsequently the quantification of each selected peptide by MS can then be undertaken (Pratt et al., 2006). Normally trypsin is used for endoproteolysis, in order to cleave proteins into peptides suitable for MS; however other endoproteinases can be used were trypsin digestion provides inappropriate surrogate peptides. Trypsin cleaves C-terminal to arginyl and lysyl residues except arginyl-propyl and lysyl-propyl sequences which are not cleaved. It produces a range of peptides between 600-4000Da which are ideal for analysis with MS. Additionally tryptic peptides are normally doubly charged ions [M+2H]2+ which aids in MS/MS analysis used in MRM experiments. Because the standard is biologically synthesised one of the advantages of QconCAT over AQUA is that once conceived and validated a QconCAT gene may be used for repeated production of unlimited amounts of isotopically-labelled peptide standards (Rivers et al., 2007). A further advantage is that were appropriate, QconCAT may be co-digested with the analyte protein in order to overcome problems associated with differential susceptibility to proteolysis (Rivers et al., 2007). Moreover it has also been suggested that by putting the peptides in sequence where they are surrounded by their native flanking sequence this may avoid this problem (Kito et al., 2007).

Experiments using AQUA and QconCAT can be performed on two MS platforms; ion trapping (e.g. Orbitrap) and quadrupole based instruments (e.g. triple quadrupole, quadrupole-time of flight (TOF)). Ion trapping instruments use AMRT. Figure 1.13 demonstrates the quadrupole methodology. For each peptide, pairs of precursor

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and fragment ion m/z values (called transitions) are monitored during the predicted elution time, allowing hundreds of peptides to be analyzed in a single experiment.

QconCAT methodology also has its shortcomings. These include the poor solubility of some QconCATs, variable expression, incomplete digestion and post translational modifications during digestion (Aebersold and Mann, 2003). In a study conducted by Mizaei et al. 2008 (Mirzaei et al., 2008) the group compared AQUA peptide and QconCAT peptide absolute quantification using the Caenorhabditis elegans proteome. Results indicated that although most QconCAT peptides were in equimolar ratios, digestion efficiencies were an important factor for some. Additionally solubilisation of some AQUA peptides was reflected in the overestimation of some QconCAT peptides. The conclusion was there was no superior method.