WO2010109022A1 - Quantitative proteomics method - Google Patents

Abstract

The present invention relates to a method for the relative or absolute quantification of analysis of two or more protein samples. The protein samples are modified to yield isobaric peptides which are subsequently submitted to tandem mass spectrometry. The method involves digestion of the protein samples and cross-wise labelling of N- and C-terminal ends of the thus obtained peptides.

Description

QUANTITATIVE PROTEOMICS METHOD

TECHNICAL FIELD

The present invention relates to the field of mass-spectrometry-based quantitative proteomics and to approaches for identifying and quantifying two or more differentially labeled states using MS/MS spectra.

BACKGROUND OF THE INVENTION Mass spectrometry (MS) is an analytical technique for the determination of the elemental composition of a sample or molecule. Jt is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios. Tandem mass spectrometry, also known as MS/MS, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.

Proteomics is the large-scale study of proteins, particularly their structures and functions.

Mass spectrometry-based quantitative proteomics involves comparison of labeled peaks. Many labeling techniques have been established which provide a distinct mass shift and allow quantitation by peak comparison on MS level (e.g. SILAC, ICAT). Relative quantitation on MS/MS level can be performed by iTRAQ using reporter ions produced during MS/MS. Nevertheless, iTRAQ requires analysis in the low mass range which is not feasible using ion traps. These techniques are further described in the below.

Accordingly, mass spectrometry-based quantitative proteomics of unknown proteins is usually performed with stable isotopic labeling or label-free approaches. Introducing stable isotopes with defined mass differences enables the comparison of different states by relative protein quantification. Stable isotope labels are most commonly introduced through metabolic and chemical approaches. Metabolic labeling techniques by e.g. stable isotope labeling by amino acids in cell culture (SILAC) requires living cells, whereas chemical labeling can be performed with any proteome. Ideally, chemical labeling requires quantitative derivatization preferably without any side reactions and is usually more successful with peptides than with proteins. Proteomics, 2005, 5(1), pp.4-15 discloses a method based on isotope-coded protein labeling (ICPL) established in intact proteins based on isotopic labeling of all amino groups using differentially deuterated forms of N- nicotinoyloxy succinimide which enabled the early incorporation of the label. Labeling on the peptide level requires that the proteomes to be compared are purified and fractionated separately which might be an additional error source for quantification. Several distinct derivatization reagents have been developed for quantitative proteomics.

Isotope-coded affinity tagging (ICAT) was the first approach for relative proteome quantification and consisting of a cysteine-directed reactive group, a linker region including different stable isotopes, and a biotin-affinity tag for purification. The ICAT- approach reduces the complexity of the sample, but is limited to cysteine-containing proteins and peptides, which is a relatively rare amino acid.

In contrast, quantitative labeling of amine-groups allowed the quantification of all peptides after proteolytic digestion and was named global internal standard technology (GIST). Stable isotopic labeling of amines can be obtained e.g. by dimethylation or succinylation with relative quantification on the MS level. Alternatively, isobaric tagging for relative and absolute quantification (iTRAQ) and tandem mass tagging (TMT) produce isobaric masses on MS level and specific reporter ions on MS/MS level for relative quantification, allowing the relative quantification of up to eight different samples simultaneously. However, TMT and iTRAQ are limited to single reporter ions in the low mass range, which are difficult to detect with e.g. ion trap detectors.

In comparison to MS/MS-based quantifications, a common feature of relative quantification by isotopic peak comparison on the MS level (e.g. SILAC, ICAT, ICPL, and GIST) is the fact that the complexity of the sample is doubled for the relative comparison of two states with increased requirements for separation by liquid chromatography (LC). On the other hand, relative isobaric quantification yielded in increased signal intensity and decreased complexity compared to MS-based quantification methods on MS/MS level using iTRAQ or TMT. Furthermore, these reagents are relatively expensive and require analysis in the low mass range (< 140 Da) which is not practical using ion trap mass spectrometers.

WO 2009/141310 discloses a method for MS analysis of analytes, which may be proteins or peptides, in which the analytes are labelled with a mass label or combination of mass labels, wherein each mass label is an isobaric mass label comprising a mass spectrometrically distinct marker group. It is mentioned that TMT, iTRAQ and iPROT may be used. WO 2009/153577 discloses a method for MS analysis of compounds, such as peptides and proteins, which have been labelled with mass markers which may be isobaric and isotopic. The peptides are labelled in the C-terminal end or in the N-terminal end. Succinic anhydride and methoxy-4,5-dihydro-1H-imidazole may be used as mass markers.

EP 1 916 526 discloses a method for MS analysis of proteins which have been labelled with a combination of an isotopic and an isobaric label. During MS a low molecular fragment ion is produced.

US 2007/0207555 describes a method for MS analysis of analytes such as proteins and peptides, which are labelled with isobaric markers. TMT, iTRAQ and iPROT may be used.

WO 2003/056299 describes compounds which are useful as labels in proteomics studies. It is mentioned that methoxy-4,5-dihydro-1 H-imidazole-d4 and methoxy-4,5-dihydro-1 H- imidazole-dO may be used as labeling compounds.

US 2003/186326 describes isotope labels for labeling of peptides to be analyzed by MS. Labelling may be achieved with succinic anhydride-dθ, succininic anhydride-d4, succinic anhydride ¹³C₀ and succinic anhydride ¹³C₄.

Hence, in view of the above, there is still a need within the art to develop new and more effective, reliable and cost-efficient quantification methods for use in MS-based methods.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an improved method for quantitative proteomics.

In accordance with the present invention there is provided a method for analysis of one or more pairs of protein samples, each pair comprising a first protein sample and a second protein sample, each pair being denoted by a pair number (i),

(i) being an integer between 1 and the total number of pairs, said method comprising the steps of

a) for at least one pair of protein samples: - separately digesting the first and the second protein sample with an enzyme to generate peptides with an amino acid at the N-terminal end and an amino acid at the C-terminal end, one of said ends is a specific amino acid;

- modifying the thus obtained peptides in the first protein sample by reaction of the amino acid at the N- or C-terminal end of the peptides with a labelling reagent denominated A^x(l) yielding peptides having an N-terminal end labelled with A^x(l) and a non-labelled C-terminal end and/or peptides having a C- terminal end labelled with A^x(l) and a non-labelled N-terminal end, followed by reaction of the non-labelled N-terminal end or C-terminal end of the peptides with a labelling reagent denominated B^y(l), yielding peptides having an N- terminal end labelled with A^x(l) and a C-terminal end labelled with B^y(l) and/or peptides having a C-terminal end labelled with A^x(l) and an N-terminal end labelled with B^y(l);

- modifying the thus obtained peptides in the second protein sample by reaction of the amino acid at the N- or C-terminal end of the peptides with a labelling reagent denominated A^y(l) yielding peptides having an N-terminal end labelled with A^y(l) and a non-labelled C-terminal end and/or peptides having a C- terminal end labelled with A^y(l) and a non-labelled N-terminal end, followed by reaction of the non-labelled N-terminal end or C-terminal end of the peptides with a labelling reagent denominated B^x(l), yielding peptides having an N- terminal end labelled with A^y(l) and a C-terminal end labelled with B^x(l) and/or peptides having a C-terminal end labelled with A^y(l) and an N-terminal end labelled with B^x(1);

b) combining the peptides obtained in step a) from at least one of said pairs of protein samples to yield a mixture of peptides, said peptides being isobaric within each pair (i) of protein samples;

c) submitting the mixture of peptides in step b) to MS/MS;

wherein x and y are stable but different isotopes, A^x(l) is a labelling reagent A containing one stable isotope x(i) for pair (i) of protein sample, A^y(l) is a labelling reagent A containing one stable isotope y(i) for pair (i) of protein sample,

B^x(l) is a labelling reagent B containing one stable isotope x(i) for pair (i) of protein sample, B^y(l) is a labelling reagent B containing one stable isotope y(i) for pair (i) of protein sample,

A^x(l), A^y(l), B^x(l) and B^y(0 may each be cleaved at their point of attachment to the peptide during MS/MS but are otherwise fragmentation resistant during MS/MS, for each pair (i) of protein sample x(i) is different from x(i) in any other pair (i) of protein sample, and for each pair (i) of protein sample y(i) is different from y(i) in any other pair (i) of protein sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Flowchart of the IPTL method. Proteins of two different states were digested with Lys-C to produce peptides with lysine residues (K) at the C-terminus. Next, the peptides were modified at lysines with MDHI and MDHI-d4, respectively. Subsequently, isobaric peptides were generated by crosswise derivatization of the N-termini with SA-d4 and SA, respectively. After mixing of the two samples, isobaric peptide masses were detected for peptides without missed cleavage sites in MS mode. Relative quantification of the two states was achieved on MS/MS level using the fragment pairs with 4 Da differences. The most significant b- and y-ions resulted in reversed quantification values.

Figure 2: MALDI-MS and MS/MS spectra of a Lys-C peptide of BSA after IPTL. The peptide mass fingerprint showed quantitative derivatization applying IPTL (Figure 2, spectrum A). In addition, MS/MS spectra of the isobaric peptide DLGEEHFK of BSA digested with Lys-C and subsequently crosswise labeled with MDHI-d4/SA and MDHI/SA- d4 are presented. The crosswise labeled peptides were mixed together in ratios 1 :1 (Figure 2, spectrum B), 2:1 (Figure 2, spectrum C) and 5:1 (Figure 2, spectrum D). The detected y-ion series occurred as doublets with 4 Da mass differences. The lower masses of these pairs corresponded to labeling with MDHI/SA-d4, and the higher masses to MDHI-d4/SA labeling. The mass range around the y2-ion at 362/366 Da was magnified to exemplify a peak pair. Neutral loss of succinic acid (yδ-SA) was observed at 1042 Da (SA- d4) and 1046 Da (SA). Figure 3: Relative quantification of standard proteins by IPTL using Mascot protein scores. Mascot scores derived from nanoLC-ESI-MS runs of three crosswise IPTL-modified Lys-C digested standard proteins (Fetuin, lactoglobulin, and transferrin) mixed at different ratios. The MS results were searched against human proteins in SwissProt using Mascot with fixed modifications of MDHI-d4/SA (gray bars) and MDHI/SA-d4 (black bars), respectively. Notably, approximately the same Mascot protein scores were achieved with the 1 :1 mixtures, whereas the 2:1 (and 1:2) mixtures revealed Mascot score ratios > 2 (and ≤ 0.5).

Figure 4: Selected MS/MS spectrum for the relative quantification of vimentin during STLC-induced apoptosis. The MS/MS spectrum of the isobaric peptide NLQEAEEWYK with m/z 741.85 digested with Lys-C and subsequently crosswise labeled with MDHI- d4/SA (STLC, 24 h) and MDHI/SA-d4 (STLC, 48 h) is presented.

Figure 5: IsobariQ-Protein view. Figure 5 shows the main window of IsobariQ where all identified proteins and their respective peptides are shown. When a peptide is quantified in the QualPLT module (figure 6) the ratio and variability of every peptide is transfered back to this protein view where the overall protein ratio and variability is calculated. This protein list can be exported to a spreadsheet application for further analysis.

Figure 6: IsobariQ - the QualPTL module. When a protein has been double-clicked in the protein view (figure 5) all MS/MS spectra assigned to this protein are displayed here. All Mascot hits assigned to a given MS/MS spectrum are displayed in the top panel (A) and the annotated MS/MS spectrum is shown in the bottom panel (B). When quantified, all the quantification events are listed in the quantification table (C) where the user can select which ratios to include or exclude for this particular MS/MS spectrum.

Figure 7A: Flowchart of IPTL using succinic anhydride with different buffers for crosswise labeling. An alternative to the original IPTL approach was developed using N-terminal succinylation for the first dervatisation step. Therefore, a specific buffer system for the succinylation reaction based on ammonium acetate was developed and applied. The second derivatisation was performed with another buffer system (sodium dihydrogen phosphate) to modify the C-terminal lysines.

Figure 7B shows N-terminal succinylation of a tryptic digest of BSA.

Figure 8A: Flowchart of tryptic-IPTL. An alternative to the original IPTL approach was developed using trypsin as enzyme (named tryptic-IPTL). Therefore, a specific buffer system for the succinylation reaction based on ammonium acetate was developed and applied. Here, the stable isotope ¹³C₄-succinic anhydride was used. The second derivatisation was performed by enzymatic incorporation of two ¹⁸O stable isotopes. ¹³C and ¹⁸O are known to have no retention time shift in contrast to deuterium.

Figure 8B: An example for the tryptic-IPTL strategy is shown. Glyceraldehyde-3- phosphate dehydrogenase from chicken was digested with trypsin and N-terminally modified with either succinic anhydride-¹³C₄ (top) or succinic anhydride (middle). Then, the sample modified with succinic anhydride was incubated with trypsin in water-O¹⁸ (bottom).

Figure 8C shows an MS/MS spectrum of a 3:1 mixture of glyceraldehyde-3-phosphate dehydrogenase from chicken.

Figure 8D shows a section of a MS/MS spectrum of a 3:1 mixture of glyceraldehyde-3- phosphate dehydrogenase from chicken. Mass pairs with 4 Da difference were found, which can be used for quantification.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for identifying and quantifying two differentially labeled states using MS/MS spectra. The method, which is hereinafter denominated isobaric peptide termini labeling (IPTL), is based on isobaric peptide termini labeling using crosswise dO and d4 reagents and relative quantification on the MS/MS level due to mass shifts of the fragment ions. In the IPTL method the proteins were digested with Lys-C to afford a peptide with a lysine residue, the produced peptides of two different states were labeled with either 2-methoxy-4,5-dihydro-1 H-imidazole (MDHI) or 2-methoxy-4,5-dihydro- 1 H-imidazole-d4 (MDHI-d4) at lysine residues following crosswise labeling with succinic anhydride-d4 (SA-d4) or SA at the N-termini. The result was a mixture of isobaric peptides being labeled at the lysine residue with MDHI and at the N-terminus with SA and/or peptides labeled at the lysine residue with MDHi-d4 and at the N-terminus with SA-d4

In the context of the present invention, also other enzymes may be used for digesting the proteins as well as other labeling agents as further exemplified herein. The term "digesting" or "digest" as mentioned herein, refers to when the proteins or peptides are divided into two or more pieces by cleavage by an enzyme which is able to perform this digestion. An endoprotease, such as Lys-C is an example of an enzyme capable of digesting proteins and/or peptides. The digestion of proteins results in peptides having a specific amino acid at one end of the peptide. For example, digestion with with Lys-C affords a peptide with a lysine residue.

In the IPTL method mixed labeled peptides without missed cleavage sites resulted in isobahc masses, with increased signal intensity and decreased complex and provided several quantification points per peptide as compared to MS-based quantitation methods. The term "isobahc peptides" refers to peptides having the same or virtually the same molecular weight. Furthermore, as referred to herein, isotopes of an element are atoms having nuclei with the same number of protons but different number of neutrons.

In contrast to e.g. iTRAQ, the present method provides more quantitation points per peptide. The IPTL method was first established with standard proteins and relative quantification was achieved by the ratio of the Mascot scores obtained by searching in both directions with fixed modifications of the two different states.

Subsequently, relative quantification using the IPTL method was further verified with the entire proteome derived from HeLa cells treated with the antimitotic inhibitor S-trityl-L- cysteine (STLC) to induce mitotic arrest after 24 h and apoptosis after 48 h as described in the experimental section. Many potentially differentially regulated proteins were identified and the majority has previously been shown to be linked to apoptosis, confirming the usefulness of the new approach of the present invention.

Accordingly, the IPTL method allows for reducing the number of peptides and increasing the number of quantification points per peptide. In this regard, the novel IPTL method is strikingly different to other MS/MS-based quantification approaches such as iTRAQ and TMT, which are limited to single reporter ions in the low mass range, which are difficult to detect with e.g. ion trap detectors. As mentioned herein, the IPTL approach was established by modification of Lys-C digested proteins with MDHI at C-terminal lysine residues and succinylation of the N-termini. The two required reagents yielded in quantitative modification. Another advantage is that the deuterated forms (d4) for isobaric labeling are commercially available. Other combinations of reagents might be applied as well, as exemplified herein, but is not limited thereto. Dimethylation of the N-termini using formaldehyde (dθ, and d2) may be used instead of succinic anhydride (dθ, and d4).

Trypsin is the most popular enzyme for proteomics applications because it produces peptides with positively charged termini of an optimal average size suited for detection by mass spectrometry. On average, Lys-C produces less and longer peptides than trypsin. This results in reduced number of peptides, particularly of complex samples, and thus considerably simplifies the requirements for LC separation and MS acquisition. Nevertheless, the IPTL method may also be performed using tryptic peptides. Labeling of arginines may be obtained using e.g, methyfglyoxal (dθ and d4, respectively), which may also be combined with N-terminal succinylation or dimethylation for crosswise isobaric labeling. Complete labeling of arginines in peptides may be difficult to achieve. On the other hand, crosswise labeling as described with MDHI and SA, would result in isobaric and isotopic tryptic peptide pairs. This approach would require combined quantification on MS/MS (single lysine-containing peptides) and MS (all other peptides) levels. Still, the number of peptides to be analyzed would be reduced and more quantification points for single lysine-containing peptides achieved in comparison to a trypsin digest without crosswise isobaric labels.

Mascot protein scores may be used for approximate relative quantification (Figure 3). Still, higher than 2-fold relative changes in protein amounts have been shown to be reliable to identify quantitative differences with standard proteins. Furthermore, the results obtained from complex protein samples of HeLa cells incubated with STLC for different lengths, revealed several similar regulated proteins as obtained by proteome analysis based on 2- DE and silver staining. Furthermore, many of the identified proteins have been found previously by other approaches to be linked to apoptosis, indicating the reliability of the novel IPTL approach. An approximate relative quantification can be obtained using the IPTL approach in combination with Mascot protein scoring. Moreover, the IPTL method may also be used for absolute quantification of proteins. Absolute quantification of proteins has been developed using stable isotope mass-tagged synthetic peptides (e.g., ¹³C and ¹⁵N) and was named AQUA. Therefore, the synthetic reference peptides serve as internal standards and are analyzed at the same time as the corresponding natural peptide by LC-MS/MS. These peptides can be analyzed by multiple selected reaction monitoring (SRM) with highest MS sensitivity. This concept has been further developed for absolute quantification of proteomes using so-called proteotypic peptides. However, the production of the stable isotope tagged synthetic peptides is relatively expensive. Alternatively, proteotyptic peptides could be produced applying the IPTL method to synthetic peptides and absolute quantification on MS/MS level.

Accordingly, the IPTL method is useful for relative quantification of proteins based on multiple fragment pairs per peptide in MS/MS mode. Quantification of isobaric peptides instead of isotopic peptides considerably reduces the number of analytes. Approximate relative quantification was achieved using Mascot protein scores. In a first aspect of the invention there is provided a method for analysis of one or more pairs of protein samples, each pair comprising a first protein sample and a second protein sample, each pair being denoted by a pair number (i),

(i) being an integer between 1 and the total number of pairs, said method comprising the steps of

a) for at least one pair of protein samples: - separately digesting the first and the second protein sample with an enzyme to generate peptides with an amino acid at the N-terminal end and an amino acid at the C-terminal end, one of said ends is a specific amino acid;

- modifying the thus obtained peptides in the first protein sample by reaction of the amino acid at the N- or C-terminal end of the peptides with a labelling reagent denominated A^x(l) yielding peptides having an N-terminal end labelled with A^x(l) and a non-labelled C-terminal end and/or peptides having a C- terminal end labelled with A^x(l) and a non-labelled N-terminal end, followed by reaction of the non-labelled N-terminal end or C-terminal end of the peptides with a labelling reagent denominated B^yW, yielding peptides having an N- terminal end labelled with A^x(l) and a C-terminal end labelled with B^y(l) and/or peptides having a C-terminal end labelled with A^x(l) and an N-terminal end labelled with B^y(l);

- modifying the thus obtained peptides in the second protein sample by reaction of the amino acid at the N- or C-terminal end of the peptides with a labelling reagent denominated A^y(l) yielding peptides having an N-terminal end labelled with A^y(l) and a non-labelled C-terminal end and/or peptides having a C- terminal end labelled with A^y(l) and a non-labelled N-terminal end, followed by reaction of the non-labelled N-terminal end or C-terminal end of the peptides with a labelling reagent denominated B^x(l), yielding peptides having an N- terminal end labelled with A^y(l) and a C-terminal end labelled with B^x(l) and/or peptides having a C-terminal end labelled with A^y(l) and an N-terminal end labelled with B^x(l); b) combining the peptides obtained in step a) from at least one of said pairs of protein samples to yield a mixture of peptides, said peptides being isobaric within each pair (i) of protein samples;

c) submitting the mixture of peptides in step b) to MS/MS;

wherein x and y are stable but different isotopes,

A^x(l) is a labelling reagent A containing one stable isotope x(i) for pair (i) of protein sample,

A^y(l) is a labelling reagent A containing one stable isotope y(i) for pair (i) of protein sample,

B^x(l) is a labelling reagent B containing one stable isotope x(i) for pair (i) of protein sample, B^y(0 is a labelling reagent B containing one stable isotope y(i) for pair (i) of protein sample,

A^x(l), A^y(l), B^x(l) and B^y(l) may each be cleaved at their point of attachment to the peptide during MS/MS but are otherwise fragmentation resistant during MS/MS, for each pair (i) of protein sample x(i) is different from x(i) in any other pair (i) of protein sample, and for each pair (i) of protein sample y(i) is different from y(i) in any other pair (i) of protein sample.

In a second aspect of the invention there is provided a method according to the first aspect of the invention, wherein the total number of pairs is equal to or less than 10.000, preferably less than 1000 and more preferably less than 100.

In a third aspect of the invention there is provided a method according to any previous aspect, wherein the MS/MS generates several quantitation points per peptide.

In a fourth aspect of the invention there is provided a method according to any previous aspect, wherein the mixture of proteins are obtained from STLC-treated HeLa cells.

In a fifth aspect of the invention there is provided a method according to any previous aspect, wherein the enzyme is an endoprotease selected from Lys-C, trypsin, Lys-N, Asp- Is!, GIu-C and Arg-C. In a sixth aspect of the invention there is provided a method according to any previous aspect, wherein reagent A^x(l) is selected from 2-methoxy-4,5-dihydro-1 H-imidazole-d4, O- methylisourea-¹³C, ¹⁵N₂, succinic anhydride-d4, succinic anhydride-¹³C₄, and water-¹⁸O.

In a seventh aspect of the invention there is provided a method according to any previous aspect, wherein reagent A^y(l) is selected from 2-methoxy-4,5-dihydro-1 H-imidazole-dO , O- methylisourea, succinic anhydride, and water.

In an 8^th aspect of the invention there is provided a method according to any previous aspect, wherein reagent B^x(l) is selected from succinic anhydride-d4 , formaldehyde-d2, propionic anhydride-¹³C₆, and succinic anhydride.

In a 9^th aspect of the invention there is provided a method according to any previous aspect, wherein reagent B^y(l) is selected from succinic anhydride-dθ , formaldehyde, propionic anhydride, succinic anhydride-d4, and succinic anhydride-¹³C₄.

In a 10^th aspect of the invention there is provided a method according to any previous aspect, wherein the enzyme is Lys-C, reagent A^x(l) is 2-methoxy-4,5-dihydro-1 H-imidazole- d4, reagent B^x(l) is succinic anhydride-d4, reagent A^y(l) is methoxy-4,5-dihydro-1 H- imidazole-dO and reagent B^y(l) is succinic anhydride -dθ.

In an 1 1^th aspect of the invention there is provided a method according to any previous aspect, wherein the method further comprises purification of the modified peptides from the first protein sample in step a) and/or purification of the modified peptides from the second protein sample in step a).

In a 12^th aspect of the invention there is provided a method according to any previous aspect, wherein the purification is selected from liquid chromatography, isoelectric focussing and gel electrophoresis.

In a 13^th aspect of the invention there is provided a method according to any previous aspect, wherein the ionization technique for MS/MS acquisition is MALDI-MS or ESI-MS.

In a 14^th aspect of the invention there is provided a method according to any previous aspect, wherein the analysis comprises identification and/or relative quantification and/or absolute quantification. In a 15^th aspect of the invention there is provided a method according to any previous aspect, wherein the ratio of the isobaric peptides from any pair (i) of protein sample is determined by the intensities of the signals from the corresponding ion fragments in the MS/MS spectrum resulting from the MS/MS acquisition.

In a 16^th aspect of the invention there is provided a method according to any previous aspect, wherein the isobaric peptides from any pair (i) of protein sample are identified using a database for protein identification.

In a 17^th aspect of the invention there is provided a method according to any previous aspect, wherein said database is the Mascot database.

In an 18^th aspect of the invention there is provided use of a method according to any previous aspect for assessment of up- or downregulation of proteins.

In a further aspect of the invention there is provided a method according to any previous aspect wherein A^x(l), A^y(l), B^x(l) and B^y(l) each have a molecular weight above 117 g/mol, 120 g/mol, 150 g/mol or 200 g/mole and may each be cleaved at their point of attachment to the peptide during MS/MS but are otherwise fragmentation resistant during MS/MS

It is to be understood that when the number of protein samples is uneven either one of the samples is discarded or one of the samples is used for forming two pairs. Additional measurements may be performed in which at least one sample is used for forming more than one pair.

All of the preceding aspects may be combined with any claims, aspects or embodiments of the invention hereinbefore or hereinafter.

FURTHER ASPECTS OF THE INVENTION

Further aspect 1. There is provided a method for comparison of two or more protein samples comprising the following steps: a) separately digesting each protein sample with an enzyme to generate peptides with a specific amino acid at the N- or C-terminal end; b) modification of the peptides from the first protein sample obtained in step a) by reaction of the specific amino acid at the N- or C-terminal end of the peptides with a stable-isotope labelled reagent A denominated A^x yielding peptides having a labelled N- or C-terminal end and a non-labelled N- or C-terminal end, c) modification of the peptides from the second protein sample obtained in step a) by reaction of the specific amino acid at the N- or C-terminal end of the peptides with a non-stable isotope labelled reagent A denominated A^y or a stable-isotope labelled reagent A denominated A^x yielding peptides having a labelled N- or C-terminal end and a non-labelled N- or C-terminal end, d) if present, modification of the further protein sample obtained in step a) by reaction of the specific amino acid at the N- or C-terminal end of the peptides with a stable-isotope labelled reagent A denominated A^x or by reaction of the specific amino acid at the N- or C-terminal end of the peptides with a non- stable-isotope labelled reagent A denominated A^y yielding peptides having a labelled N- or C-terminal end and a non-labelled N- or C-terminal end; e) combining the peptides obtained in steps b), c) and/or d) to give a mixture of isobaric peptides; f) analysis of the mixture of isobaric peptides obtained in step e).

Further aspect 2. There is provided a method according to further aspect 1 of the invention wherein the peptides from the first protein sample in step b) are further modified by reaction of the specific amino acid of the non-labelled N-or C-terminal end of the peptides with a non-stable-isotope labelled reagent B denominated B^y or with a stable- isotope labelled reagent denominated B^x.

Further aspect 3. There is provided a method according to any previous further aspect wherein the peptides from the second protein sample in step c) are further modified by reaction of the amino acid of the non-labelled N-or C-terminal end of the peptides with a stable-isotope labelled reagent B denominated B^x or with a non-stable-isotope labelled reagent denominated B^y.

Further aspect 4. There is provided a method according to any previous further aspect wherein the peptides from the further protein sample in step d) are further modified by reaction of the amino acid of the non-labelled N-or C-terminal end of the peptides with a non-stable-isotope labelled reagent B denominated B^y or by reaction of the amino acid of the non-labelled N-or C-terminal end of the peptides with a stable-isotope labelled reagent B denominated B^x. Further aspect 5. There is provided a method according to any previous further aspect wherein the comparison comprises identification and/or relative quantification and/or absolute quantification.

Further aspect 6. There is provided a method according to any previous further aspect wherein the method further comprises purification after one or more of steps a); b); c) and d).

Further aspect 7. There is provided a method according to any previous further aspect wherein the enzyme is an endoprotease such as Lys-C, trypsin, Lys-N, Asp-N, GIu-C and Arg-C.

Further aspect 8. There is provided a method according to any previous further aspect wherein reagent A^y is selected from MDHI.

Further aspect 9. There is provided a method according to any previous further aspect wherein reagent B is selected from SA or formaldehyde.

Further aspect 10. There is provided a method according to any previous further aspect wherein the enzyme is Lys-C, reagent A^x is MDHI-d4, reagent B^y is SA, reagent A^y is MDHI and reagent B^x is SA-d4.

Further aspect 11. There is provided a method according to any previous further aspect wherein the protein samples independently comprise the same proteins or a mixture of different proteins.

Further aspect 12. There is provided a method according to any previous further aspect wherein the mixture of different proteins are obtained from STLC-treated HeLa cells.

Further aspect 13. There is provided a method according to any previous further aspect wherein the mixture of isobaric peptides is analyzed using MS/MS acquisition.

Further aspect 14. There is provided a method according to any previous further aspect wherein the combined protein sample is subjected to liquid chromatography prior to MS/MS acquisition. Further aspect 15. There is provided a method according to any previous further aspect wherein the MS/MS acquisition is MALDI-MS and ESI-MS.

Further aspect 16. There is provided a method according to any previous further aspect wherein the ratio of the isobaric peptides from the first, second and/or further protein samples is determined by the intensities of the signals from the corresponding stable isotope-labelled and/or non-stable isotope-labelled ion fragments in the MS/MS spectrum resulting from the MS/MS acquisition.

Further aspect 17. There is provided a method according to any previous further aspect wherein the isobaric peptides are identified using a database for protein identification.

Further aspect 18. There is provided a method according to any previous further aspect wherein said database is the Mascot database.

Further aspect 19. There is provided a use of a method according to any previous further aspect for assessment of up- or downregulation of proteins wherein the isobaric peptides are identified using a database for protein identification.

The invention is illustrated, but not limited, by the following examples.

EXAMPLES Cell culture, induction of apoptosis, and SDS-PAGE.

HeLa cells were grown as a monolayer in RPMI supplemented with 10% foetal bovine serum and maintained in a humid incubator at 37°C in a 5% CO₂ environment. Cells were treated with 5 μM S-trityl-L-cysteine (STLC) from a 5 mg/mL stock in DMSO. Cells were trypsinized after 24 h, and 48 h, harvested, resuspended in 1 ml PBS and centrifuged again at 10.000 rpm. Cell pellets were frozen in liquid nitrogen and stored at -20⁰C.

SDS-PAGE was performed with 4% stacking gel and 10% separation gel using a Mini- Protean 3 cell (Bio-Rad, Oslo, Norway). Gels were stained with Coomassie Brilliant Blue G-250 (Serva, Heidelberg, Germany) employing the blue silver staining technique with slight modifications. Fixation was performed with 50% ethanol/2% phosphoric acid for 1 h, incubation with 34% ethanol/2% phosphoric acid/17% ammonium sulfate for 1 h, and staining with 20% methanol/10% phosphoric acid/10% ammonium sulfate for 1 h. Finally, the gels were washed once for 30 min with 25% ethanol and three times with water.

In-gel Lys-C digestion. Coomassie G-250 stained gel lanes were cut into 12 bands with a scalpel for in-gel digestion with 0.06 μg Lys-C (Sigma-Aldrich, Oslo, Norway) in 60 μL 25 mM Tris pH 8, 1 mM EDTA for 16 h at 37°C. For each band the Lys-C produced peptides were purified with μ-C18 ZipTips (Millipore, Billerica, MA, USA), and dried using a Speed Vac concentrator (Savant, Holbrook, NY, USA).

In-solution Lys C digestion.

Fetuin (bovine), lactoglobulin (bovine), transferrin (human), and serum albumin (bovine) were purchased form Sigma-Aldrich (Oslo, Norway). The proteins were dissolved in Lys-C buffer (25 mM Tris pH 8.5, and 1 mM EDTA) and digested with Lys-C (enzyme to protein ratio 1 :50 ) for 16 h at 37°C. The digestion was stopped by adding formic acid to a final concentration of 0.8%.

Derivatization of lysine residues.

2-Methoxy-4,5-dihydro-1 H-imidazole (MDHI) and the deuterated form 2-methoxy-4,5- dihydro-1 H-imidazol-4,4,5,5-d4 (MDHI-d4) of the reagent were purchased from C/D/N Isotopes (Point Claire, Quebec, Canada). An aqueous solution of 800 mM 2-methoxy-4,5- dihydro-1 H-imidazole (dθ and d4) was prepared and 10 μl of this solution was added to the purified and dried Lys-C digests dissolved in 10 μl water/acetonitrile/trifluoroacetic acid (89.9:10:0.1 v/v/v), and incubated for 3 h at 55 ⁰C. The reaction was stopped with 1 μl trifluoroacetic acid and the Lys-C peptides were purified with μ-C18 ZipTips (Millipore, Billerica, MA, USA), and dried using a Speed Vac concentrator (Savant, Holbrook, NY, USA).

N-terminal peptide succinylation. A solution of 100 mM succinic anhydride (SA) (Sigma-Aldrich, Oslo, Norway) or succinic anhydride-d4 (SA-d4) (Larodan Fine Chemicals AB, Malmδ, Sweden) was freshly prepared in 200 mM sodium dihydrogenphosphate buffer and the pH was adjusted to 6.5 with ammonium hydroxide. 10 μl of SA-solution was added to the purified and dried Lys-C peptide digests derivatized with MDHI-d4 or 10 μl of SA-d4-solution was added to the dried Lys-C peptide digests derivatized with MDHI. After incubation for 2 h at 37°C and the reaction was stopped with 1 μl 1% trifluoroacetic acid, purified with μ-C18 ZipTips (Millipore, Billerica, MA, USA), and dried using a Speed Vac concentrator (Savant, Holbrook, NY, USA). For analysis by mass spectrometry, the modified Lys-C peptides were reconstituted in 5 μl 1% formic acid and combined.

MALDI-TOF/TOF-MS. An Ultraflex Il (Bruker Daltonics, Bremen, Germany) MALDI-TOF/TOF mass spectrometer was used with a mass accuracy of 50 ppm after external calibration with kemptide, bradykinin, substance P, glu-fibrinopeptide B, and dynorphin A 2-17 (Sigma-AIdrich, Oslo, Norway or Bachem, Basel, Switzerland). The samples were analyzed in the MS mode for the generation of peptide mass fingerprints as well as in the TOF/TOF mode for fragmentation analysis of chosen peaks. 4-Hydroxycinnamic acid (20 mg/mL) in 0.3% aqueous trifluoroacetic acid/acetonitrile (2:1) was used as matrix. The samples were applied to a ground steel sample holder and introduced into the mass spectrometer after drying. Basic settings of the MALDI-TOF/TOF instrument (Ultraflex II, Bruker Daltonics, Bremen, Germany) were as follows: Ion source 1 , 25 kV; ion source 2: 21.85 kV; lens: 9.60 kV; reflector: 26.3 kV; reflector 2, 13.85 kV; deflector mode, polarity positive. Mass spectra were transformed into peak lists by using the SNAP algorithm of the software FlexAnalysis version 2.4 (Bruker Daltonics, Bremen, Germany).

Nano-LC-LTQ Orbitrap mass spectrometry. The dried peptides were dissolved in 10 μl 1% formic acid in water and 3 μl were injected onto an LC/MS system consisting of an Ultimate 3000 nano-LC system (Dionex, Sunnyvale CA, USA) connected to a linear quadrupole ion trap-orbitrap (LTQ Orbitrap XL) mass spectrometer (ThermoScientific, Bremen, Germany) equipped with a nanoelectrospray ion source. An Acclaim PepMap 100 column (C18, 3 μm, 100 A) (Dionex, Sunnyvale CA, USA) with a capillary of 12 cm bed length was used for separation by liquid chromatography. The flow rate used was 300 nL/min for the nano column, and the solvent gradient used was 7% B to 50% B in 45 minutes. Solvent A was 0.1% formic acid, whereas aqueous 90% acetonitrile in 0.1 % formic acid was used as solvent B. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition. Survey full scan MS spectra (from m/z 300 to 2,000) were acquired in the Orbitrap with resolution R = 60,000 at m/z 400 (after accumulation to a target of 1 ,000,000 charges in the LTQ). The method used allowed sequential isolation of the most intense ions, up to six, depending on signal intensity, for fragmentation on the linear ion trap using collisionally induced dissociation at a target value of 100,000 charges. For accurate mass measurements the lock mass option was enabled in MS mode and the polydimethyicyclosiloxane (PCM) ions generated in the electrospray process from ambient air were used for internal recalibration during the analysis. Target ions already selected for MS/MS were dynamically excluded for 60 seconds. General mass spectrometry conditions were: electrospray voltage, 1.5 kV; no sheath and auxiliary gas flow. Ion selection threshold was 500 counts for MS/MS, and an activation Q-value of 0.25 and activation time of 30 ms were also applied for MS/MS.

Data analysis. Raw LTQ Orbitrap XL data were processed using DTA supercharge software to generate mgf files. Then, a database search was performed by tandem mass spectrometry ion search algorithms from the Mascot in-house version 2.2.1 by database comparisons with mammalian (63892 sequences) or human entries (20411 sequences) from Swiss-Prot (20081212). Lys-C was selected as enzyme without any missed cleavage sites and tolerance of 10 ppm for the precursor ion and 0.6 Da for the MS/MS fragments was applied. Moreover, methionine oxidation was allowed as variable modification. Fixed modifications were set to the two corresponding modifications SA/MDHI-d4 or SA- d4/MDHI, respectively. Automatic decoy database searches were performed in Mascot and revealed a false discovery rate for peptide matches above an identity threshold of less than 2%. Proteins were considered to be identified by Mascot if a probability < 0.01 was achieved. "Show sub-sets" and "require bold red" were applied on initial Mascot search results to eliminate redundancy. Approximate relative quantification was achieved by comparison of the ratios of the Mascot protein scores using SA/MDHI-d4 and SA- d4/MDHI as fixed modifications.

Results

Strategy for quantitative proteomics using isobaric peptide termini labeling (IPTL). Crosswise peptide termini labeling was performed to produce isobaric peptides to compare two different states of a protein sample and to distinguish them after mass spectrometry acquisition (Figure 1). First, the proteins were digested with endoprotease Lys-C to generate peptides with lysines at the C-terminal end. The free lysines were subsequently modified with MDHI-d4 (state A), and MDHI (state B), respectively. The second chemical modification was carried out to modify the N-termini with succinic anhydride. Here, the peptides from state A were labeled with SA whereas the peptides from state B were modified with the SA-d4. The labeled peptides with single lysines from both combined states resulted in isobaric masses with identical physico-chemical properties. Moreover, isobaric peptides co-eluted during reverse phase LC-separation and were selected for MS/MS acquisition at the same time Therefore, corresponding peptides of the two states resulted in single masses in MS mode The relative quantitative abundance of the peptides deriving from the two different states can be detected by the ion intensities in the MS/MS spectrum, occurring in pairs with 4 Da mass shifts Here, the b-ιons of the peak pairs with lower masses (dθ) derived from proteins state A and the higher masses (d4) from proteins state B and vice versa for the y-ιons (Figure 1)

Analysis of IPTL-deπvatised BSA

To assess the feasibility of the IPTL approach, we mixed two differentially labelled endoproteinase Lys C digests of BSA (MDHI/SA-d4 and MDHI-d4/SA) in the ratios 1 1 , 1 2 and 1 5 When the resulting peptide mass fingerprint was analysed by MALDJ-TOF MS, all the major peaks in the spectrum could be assigned to IPTL-labelled BSA-peptides indicating that both denvatisation reactions had run to completion (Fig 2A) Analysis of this digest by LCMSMS yielded spectra with peak doublets that were separated by 4 Da Most of these peaks could be assigned to y-ιons and peak heights and peak areas correlated very well with the ratios of the mixtures (Fig 2B, Table 1) The spectrum in Fig 2B and the data in table 1 illustrate one important feature if IPTL, namely that every peptides generates many datapoints that are suitable for quantification, enabling the use of statistical methods to assess the confidence of a quantification event and reducing the impact of potentially interfering fragment ions

Mascot score ratios as a quick estimate of protein abundance

We wanted to use IPTL to assess quickly and with high confidence, which proteins are either upregulated or downregulated in response to chemically induced apoptosis To do this without resorting to the development of a tailored spectral processing program, we took advantage of one particular feature of the Mascot peptide identification algorithm Non-matched peaks in an MSMS spectrum incur a penalty in the Mascot score that increases with the intensity of these non-matched peaks When a protein database is searched with MDHI and SA-d4 as constant peptide modifications on Lys and on the C- terminus, respectively, peaks that result from MDHI-d4 and SA labelling will not be matched to a sequence and vice versa As a result, we anticipated that the score for MDHI-d4/SA labelled peptides would be much higher when these peptides are relatively more abundant To test this hypothesis a digest of fetuin, lactoglobulin and transferrin that were labelled with MDHI-d4/SA or with MDHI/SA-d4 in 9 different rations (5 1 , 4 1 , 3 1 , 2 1 , 1 1 , 1 2, 1 3, 1 4 and 1 5) was analysed by nano-LC-ESI-MSMS and the data were searched using Mascot setting either MDHI-d4/SA or MDHI/SA-d4 as fixed modifications for the peptide N-termini and Lys residues. We were surprised to find that the Mascot score ratios corresponded almost linearly to the ratios of the protein mixtures (Fig. 3).

Quantitative proteome analysis of STLC-induced apoptosis using IPTL After establishing and vehfiying our novel approach with standard proteins, we wanted to confirm its usefulness on the cellular proteome level. We therefore chose to compare HeLa cells incubated during different time length with the well characterized antimitotic inhibitor S-trityl-L-cysteine (STLC). STLC is a reversible inhibitor of kinesin Eg5 and inhibits tumor growth. STLC blocks cells in the M phase of the cell cycle and subsequently leads to apoptosis. HeLa cells treated with 5 μM STLC for 24 h to induce mitotic arrest and 48 h to trigger apoptosis and were lysed and the proteins from these lysates were separated by SDS-PAGE. Each lane was cut into 12 slices, proteins were in gel digested and the peptides from corresponding slices were either labelled with MDHI-d4/SA (24 h STLC treatment) or with MDHI/SA-d4 (48 h treatment). Peptides from corresponding slices were pooled and analysed by LCMS. The resulting MS data were searched against the SwissProt database using Mascot setting either MDHI-d4/SA or MDHI/SA-d4 as constant modifications.

In total, 408 proteins were identified with p<0.01. After excluding proteins that were identified with single peptides from quantification, 136 proteins were identified in both searches with a median Mascot score ratio of 1.00 (supplementary table 1). 19 proteins returned a Mascot score ratio > 2 and 28 proteins returned a Mascot score ratio of < 0.5 with a minimum of three matched queries and a Mascot score of at least 50 in one of the two searches. An additional 30 proteins were not identified at all when searched with MDHI-d4/SA as fixed modification despite being identified with at least 3 matched peptides and a Mascot score of > 50 when MDHI when MDHI/SA-d4 was set as fixed modifications. Conversely, 14 proteins were only identified when MDHI-d4/SA was set as the fixed modifications (with at least three peptides and a Mascot score > 50), but not when MDHI/SA-d4 was set as the fixed modifications. Many of the proteins that were highlighted by this analysis were detected in a previous, two-dimensional electrophoresis- based study of STLC-induced apoptosis or in analyses of apoptotic events reported by other laboratories (Table 1). One of these, vimentin, was identified with three matched peptides and Mascot protein scores of 97 and 33, respectively, resulting in a Mascot score ratio of 2.94. Analysis of the MSMS spectrum of one of these three peptides, NLQEAEEWYK revealed almost exactly this ratio when the peak heights of the matching b- and y-ions are compared (Fig. 4). Table 1 STLC-modified proteins identified using IPTL

The protein names, Swiss Prot accession numbers, the Mascot protein score (Score) and the number of matched queries (QM) searching the respective set of fixed modifications are displayed Incubation of HeIa cells with STLC for 24 h to induce mitotic arrest corresponded to MDHI-d4/SA and for 48 h to trigger apoptosis to MDHI/SA-d4 The relative protein score (ReI score) was calculated if the protein was identified with both sets of fixed modifications (MDHI-d4/SA)/(MDHI/SA-d4) Most of the proteins have previously been identified to be linked to apoptosis

Protein name Ace. no Score/QM Score/QM ReI.

(MDHI-d4/ (MDHI/ score SA)- SA-d4)

Polyadenylate-binding protein 4 Q13310 110/9 29/3 3,79

Nucleolin P19338 415/32 112/14 3,71

T-complex protein 1 subunit eta Q99832 117/7 33/4 3,55

Heat shock protein 105 kDa Q92598 123/12 35/5 3,51

Stress-70 protein, mitochondrial P38646 114/7 38/3 3,00

Vimentin P08670 97/3 33/3 2,94

Pyruvate kinase isozymes M1/M2 P14618 132/14 46/8 2,87

Histone H1 2 P16403 169/11 59/4 2,86

Lamin-A/C P02545 170/9 61/6 2,79

T-complex protein 1 subunit beta P78371 140/7 52/7 2,69

Purine nucleoside phosphorylase P00491 85/6 33/1 2,58

Histone-binding protein RBBP4 Q09028 74/6 29/2 2,55

Heat shock cognate 71 kDa protein P11142 652/50 278/28 2,35

T-complex protein 1 subunit delta P50991 63/6 27/2 2,33

U2 small nuclear πbonucleoprotein A' P09661 62/4 27/1 2,30

Nuclear autoantigenic sperm protein P49321 80/4 35/1 2,29

Far upstream element-binding protein 2 Q92945 54/5 25/2 2 16

Protein disulfide-isomerase P07237 135/8 63/5 2,14

Catalase P04040 60/3 28/1 2,14

Protein RCC2 Q9P258 41/1 82/4 0,50

Thioredoxin P10599 50/2 101/4 0,50

Malate dehydrogenase, mitochondrial P40926 35/3 73/4 0,48

3-hydroxyιsobutyryl-CoA hydrolase, mitochondrial Q6NVY1 34/1 71/3 0,48 lnosιne-5'-monophosphate dehydrogenase 2 P12268 51/6 107/7 0,48

Fructose-bisphosphate aldolase A P04075 213/11 462/23 0,46

L-lactate dehydrogenase B chain P07195 46/4 103/6 0,45

Kιnesιn-1 heavy chain P33176 36/3 81/8 0,44

Poly ⁴¹ polymerase 1 P09874 41/2 93/4 0,44

3-ketoacyl-CoA thiolase, mitochondrial P42765 3613 84/6 0,43

Stathmin P16949 25/1 59/5 0,42

Annexin A1 P04083 38/5 94/7 0,40

60S ribosomal protein L4 P36578 51/2 133/6 0,38

Nucleophosmin P06748 58/5 154/19 0,38 Medium-chain specific acyl-CoA dehydrogenase P11310 33/1 90/3 0,37

60 kDa heat shock protein, mitochondrial P10809 388/34 1064/52 0,36

Talιn-1 Q9Y490 29/1 101/5 0,29

Alpha-actιnιn-4 043707 34/3 131/12 0,26

RuvB-like 1 Q9Y265 28/1 117/5 0,24

Phosphoglycerate kinase 1 P00558 38/2 165/19 0,23

Annexin A2 P07355 39/5 173/14 0,23

Serpin H1 P50454 39/3 176/9 0,22 lnterleukin enhancer-binding factor 3 Q12906 36/6 163/9 0,22

Actin, cytoplasmic 1 P60709 55/6 316/35 0,17

Filamin-B 075369 26/2 159/10 0,16

Histone H4 P62805 25/1 203/4 0,12

Alpha-enolase P06733 34/6 311/17 0,11

Myosιn-9 P35579 4614 734/31 0,06

Serine/threonine-protein kinase PAK 2 Q13177 169/10

S-methyl-5'-thιoadenosιne phosphorylase Q13126 111/4

Caldesmon Q05682 87/3

Src substrate cortactin Q 14247 81/4

ATP-dependent DNA helicase 2 subunit 2 P13010 77/7

26S proteasome non-ATPase regulatory subunit 3 043242 77/3

Heterogeneous nuclear πbonucleoprotein IVl P52272 72/4

Ubiquitin-like modifier-activating enzyme 1 P22314 69/4

Targeting protein for XkI⁴⁷ Q9ULW0 68/3

Heterogeneous nuclear πbonucleoprotein Q 060506 62/5

Putative adenosylhomocysteinase 2 043865 62/3

Proteasome subunit alpha type-1 P25786 61/3

Lysyl-tRNA synthetase Q15046 58/3

ATP-binding cassette sub-family E member 1 P61221 57/4

GTP-binding nuclear protein Ran P62826 417/13

Elongation factor 1 -alpha 1 P68104 210/10

Peroxιredoxιn-1 Q06830 121/16

Peroxιredoxιn-2 P32119 115/4

ATP synthase subunit beta, mitochondrial P06576 113/3

Protein DJ-1 Q99497 111/4

Transgelιn-2 P37802 111/6

Ras-related protein Rab-7a P51149 108/3

Heterogeneous nuclear ribonucleoprotein U Q00839 101/8

Histone H2B type 1-C/E/F/G/l P62807 98/4

Nucleoprotein TPR P12270 86/6

Elongation factor 1 -gamma P26641 84/5

Alpha-actιnιn-1 P12814 82/6

6-phosphogluconate dehydrogenase, decarboxyl P52209 79/5

Puromycin-sensitive aminopeptidase P55786 78/3

Catenin alpha-1 P35221 77/6

Eukaryotic translation initiation factor 4 gamma 1 Q04637 73/3

Fumarate hydratase, mitochondrial P07954 68/5 RuvB-like 2 Q9Y230 63/3

Spectrin alpha chain Q13813 62/3

Glyoxalase domain-containing protein 4 Q9HC38 61/3

Plectιn-1 Q15149 58/5

Adenylyl cyclase-associated protein 1 Q01518 55/3

High mobility group protein B2 P26583 55/5

4OS ribosomal protein S7 P62081 55/3

Hepatoma-deπved growth factor P51858 53/6

Histone H2B type 1-B P33778 51/3

PEST proteolytic signal-containing nuclear protein Q8WW12 51/3

Leukotπene A-4 hydrolase P09960 50/3

Acyl-protein thioesterase 1 075608 50/4

A software solution for the IPTL method. Procedure for protein identification using Mascot and quantification using IsobariQ 1 Process raw data to mgf files Mgf-files can be generated using different software depending on the instrument used and the type of raw data For Thermo instruments we recommend using DTASuperCharge available free of charge from http //msquant alwaysdata net/msq/download/

2 Perform a database search with Mascot using following search parameters Lys-C as enzyme with no missed cleavage sites, N-terminal protein acetylation, methionine oxidation, SA, SA-d4, MDHI, MDHI-d4 as variable modifications No fixed modifications, and apply the automatic decoy database to determine the false discovery rate The mass accuracies are dependent on the instrument used

3 Copy the Mascot result file (dat-file) to a local folder 4 Launch IsobariQ and from the file menu select "Open Mascot dat-file" In the dialog select the correct dat-file and click "open" IsobariQ processes the Mascot dat-file and displays the Mascot results in a table (figure 5) where every protein can be clicked to display its assigned peptides and their individual scores IsobariQ is not limited to Mascot as search engine because it can also read separate mgf-files with corresponding identifications in a pepXML file Currently, IsobariQ is only available as a test version (v 0 1)

5 Double clicking on a protein loads this protein with its assigned peptides and MS/MS spectra into the quantification module QualPTL (figure 6) The first MS/MS spectrum identifying this protein is shown in the bottom panel and all Mascot hits to this MS/MS spectrum are shown in the top panel with detailed information about sequence, ions score, modifications and ppm error (figure 6A) This is the same information as given by Mascot when hovering over a query number with the mouse in the web-view. The spectrum updates its annotations accordingly by clicking on different sequence annotations (figure 6B). For IPTL-labeled peptides, two identifications for the same peptide should be found: One with the modifications SA/MDHI-d4 and the other with SA-d4/MDHI. As a control, clicking on these two should change the b- and y-ion series annotation from light to heavy and vice versa.

6. Clicking on the "Quantitate"-button informs QualPTL to detect all ion pairs which have been assigned to a sequence fragment, and calculate the individual ratios of these. The results are shown in the quantification table (figure 6C). In this table the user may select which ratios to include or exclude in the final quantification of this peptide.

7. Once a peptide is quantified the ratio and variability of the peptide is transferred back to the protein table where also the overall protein ratio and variability is calculated. For further analysis the protein list and all its quantification information can be exported to a spreadsheet application via a tab-separated values (tsv) file. From the file menu choose "Save As.." and give the file a unique name ending with .tsv. In the spreadsheet application the protein list can be opened and processed for post-quantification analysis like ratio normalization and significance determination.

Abbreviations

GIST, global internal standard technology; ICAT, isotope-coded affinity tagging; ICPL. isotope-coded protein labeling; IPTL, isobaric peptide termini labeling; iTRAQ, isobaric tagging for relative and absolute quantification; MDHI, methoxy-4,5-dihydro-1 H-irnidazole;

SA, succinic anhydride; SILAC, stable isotope labeling by amino acids in cell culture; STLC, S-trityl-L-cysteine; TMT, Tandem mass tagging.

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

DMSO: dimethyl sulfoxide

EDTA: ethylenediaminetetraacetic acid

LTQ: linear trap quadrupole MALDI/TOF MS : matrix assisted laser desorption ionization / time of flight mass spectroscopy

PCM: polydimethylcyclosiloxane

BSA Bovine Serum Albumine

Claims

CLAIMS 1. A method for analysis of one or more pairs of protein samples, each pair comprising a first protein sample and a second protein sample, each pair being denoted by a pair number (i), (i) being an integer between 1 and the total number of pairs, said method comprising the steps of

a) for at least one pair of protein samples:

- separately digesting the first and the second protein sample with an enzyme to generate peptides with an amino acid at the N-terminal end and an amino acid at the C-terminal end, one of said ends is a specific amino acid;

- modifying the thus obtained peptides in the first protein sample by reaction of the amino acid at the N- or C-terminal end of the peptides with a labelling reagent denominated A^x(l) yielding peptides having an N-terminal end labelled with A^x(l) and a non-labelled C-terminal end and/or peptides having a C- terminal end labelled with A^x(l) and a non-labelled N-terminal end, followed by reaction of the non-labelled N-terminal end or C-terminal end of the peptides with a labelling reagent denominated B^y(l), yielding peptides having an N- terminal end labelled with A^x(l) and a C-terminal end labelled with B^y(l) and/or peptides having a C-terminal end labelled with A^x(l) and an N-terminal end labelled with B^y(l);

- modifying the thus obtained peptides in the second protein sample by reaction of the amino acid at the N- or C-terminal end of the peptides with a labelling reagent denominated A^y(l) yielding peptides having an N-terminal end labelled with A^y(l) and a non-labelled C-terminal end and/or peptides having a C- terminal end labelled with A^y(l) and a non-labelled N-terminal end, followed by reaction of the non-labelled N-terminal end or C-terminal end of the peptides with a labelling reagent denominated B^x(l), yielding peptides having an N- terminal end labelled with A^y(l) and a C-terminal end labelled with B^x(l) and/or peptides having a C-terminal end labelled with A^y(l) and an N-terminal end labelled with B^x(l);

b) combining the peptides obtained in step a) from at least one of said pairs of protein samples to yield a mixture of peptides, said peptides being isobaric within each pair (i) of protein samples; c) submitting the mixture of peptides in step b) to MS/MS;

wherein x and y are stable but different isotopes,

A^x(l) is a labelling reagent A containing one stable isotope x(i) for pair (i) of protein sample,

A^y(l) is a labelling reagent A containing one stable isotope y(i) for pair (i) of protein sample, B^x(l) is a labelling reagent B containing one stable isotope x(i) for pair (i) of protein sample,

B^y(l) is a labelling reagent B containing one stable isotope y(i) for pair (i) of protein sample,

A^x(l), A^y(l), B^x(l) and B^y(l) may each be cleaved at their point of attachment to the peptide during MS/MS but are otherwise fragmentation resistant during MS/MS, for each pair (i) of protein sample x(i) is different from x(i) in any other pair (i) of protein sample, and for each pair (i) of protein sample y(i) is different from y(i) in any other pair (i) of protein sample.

2. A method according to claim 1 , wherein the total number of pairs is equal to or less than 10.000, preferably less than 1000 and more preferably less than 100.

3. A method according to any previous claim, wherein the MS/MS generates several quantitation points per peptide.

4. A method according to any previous claim, wherein the mixture of proteins are obtained from STLC-treated HeLa cells.

5. A method according to any previous claim, wherein the enzyme is an endoprotease selected from Lys-C, trypsin, Lys-N, Asp-N, GIu-C and Arg-C.

6. A method according to any previous claim, wherein reagent A^m is selected from 2- methoxy-4,5-dihydro-1 H-imidazole-d4, O-methylisourea-¹³C, ¹⁵N₂, succinic anhydride-d4, succinic anhydride-¹³C₄, and water-¹⁸O.

7. A method according to any previous claim, wherein reagent A^y(l) is selected from 2- methoxy-4,5-dihydro-1 H-imidazole-dO , O-methylisourea, succinic anhydride, and water.

8. A method according to any previous claim, wherein reagent B^x(l) is selected from succinic anhydride-d4 , formaldehyde-d2, propionic anhydride-¹³C₆, and succinic anhydride.

9. A method according to any previous claim, wherein reagent B^y(l) is selected from succinic anhydride-dO , formaldehyde, propionic anhydride, succinic anhydride-d4, and succinic anhydride-¹³C₄.

10. A method according to any previous claim, wherein the enzyme is Lys-C, reagent A^x(l) is 2-methoxy-4,5-dihydro-1 H-imidazole-d4, reagent B^x(l) is succinic anhydride- d4, reagent A^y(l) is methoxy-4,5-dihydro-1H-imidazole-dO and reagent B^y(l) is succinic anhydride -dθ.

1 1. A method according to any previous claim, wherein the method further comprises purification of the modified peptides from the first protein sample in step a) and/or purification of the modified peptides from the second protein sample in step a).

12. A method according to any previous claim, wherein the purification is selected from liquid chromatography, isoelectric focussing and gel electrophoresis.

13. A method according to any previous claim, wherein the ionization technique for

MS/MS acquisition is MALDI-MS or ESI-MS.

14. A method according to any previous claim, wherein the analysis comprises identification and/or relative quantification and/or absolute quantification.

15. A method according to any previous claim, wherein the ratio of the isobaric peptides from any pair (i) of protein sample is determined by the intensities of the signals from the corresponding ion fragments in the MS/MS spectrum resulting from the MS/MS acquisition.

16. A method according to any previous claim, wherein the isobaric peptides from any pair (i) of protein sample are identified using a database for protein identification.

17. A method according to any previous claim, wherein said database is the Mascot database.

18. Use of a method according to any previous claim for assessment of up- or downregulation of proteins.