Selective Isolation of Lysine-Free Tryptic Peptides Delimited by

Selective Isolation of Lysine-Free Tryptic Peptides Delimited by Arginine Residues: A New Tool for Proteome Analysis Aniel Sa´ nchez,† Luis J. Gonza´ lez,*,† Yassel Ramos,† La´ zaro Betancourt,† Jeovanis Gil,† Vladimir Besada,† Jorge Ferna´ ndez-de-Cossio,‡ Fe´ lix Alvarez,† and Gabriel Padro´ n† Mass Spectrometry Laboratory, Department of Proteomics, Department of Informatics, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba Received January 4, 2006

Tryptic digestion of biotinylated Lys-C peptides followed by affinity chromatography allows the selective isolation of lysine-free tryptic peptides delimited by arginine residues (RRnK peptides). In silico analysis revealed that RRnK peptides represent 87% of the whole proteomes and their specific isolation simplifies the complex peptide mixture (5 peptides per protein). The good recoveries and high selectivity obtained in the isolation of RRnK peptides anticipate the applicability of this method in 2DE-free quantitative proteome analyses. Keywords: proteomics • selective-peptide-isolation • protein-identification • affinity chromatography • mass spectrometry

Introduction Classical proteome analysis comprises the separation of a complex mixture of proteins by two-dimensional gel electrophoresis (2DE), followed by image analysis in order to determine differentially expressed proteins and their identification by mass spectrometry. Although 2DE is the most resolving technique for the analysis of very complex protein mixtures,1,2 it has inherent limitations such its laboriousness and difficulties for full automation,3 which seriously compromises a reasonable high-throughput. Particularly, its limited loading capacity and sensitivity restrict the detection of low-abundant proteins4 which represents a considerable and important portion of the proteomes. Furthermore, despite considerable advances in this field, the analysis of membrane proteins5 by 2DE remains a challenge. For all these reasons, one trend has been the characterization of proteolytic peptides as surrogates of the proteins present in the cell lysate by using the combination of multidimensional liquid chromatography and mass spectrometry. In this direction, a very efficient method known as MudPiT has been developed.6;7 However, due to insufficient capabilities of current mass spectrometers and chromatography systems, it has become evident the necessity of a previous step that decreases the complexity of these mixtures of peptides while, at the same time, preserving enough information to perform a quantitative analysis of as many proteins as possible,8 including lowabundant proteins. In this sense, several approaches based on the selective isolation of proteolytic peptides sharing common features in * To whom correspondence should be addressed. Mass Spectrometry Laboratory, Department of Proteomics, Division of Physical-Chemistry, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba. Fax: (53-7) 2714764, E-mail: [email protected]. † Mass Spectrometry Laboratory, Department of Proteomics. ‡ Department of Informatics.

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Published on Web 03/28/2006

their sequences and representing the individual proteins in the analyzed mixture have been developed. A pioneering work in this field was introduced by Aebersold and co-workers9,10 when they developed the methodology known as ICAT (Isotope Coded Affinity Tag). In the two compared conditions, the side chain of cysteine residues is modified using a light- and a heavy-isotope-labeled biotin tag, respectively. This procedure not only allows the selective isolation of a subset of cysteine containing peptides by affinity chromatography but also measures the relative amount of the proteins expressed in the compared conditions.11-13 Using this technique, it is possible to analyze approximately 90% of the proteomes under study while, at the same time, a reduced number of cysteine containing peptides (average 3-4 peptides/ protein) are selectively isolated reducing the complexity of the peptide mixture. To accomplish similar purposes, other chromatographic systems have been used that allow the selective isolation of chemically modified peptides in other low-frequent amino acids such as methionine,14 tryptophan,15,16 and serine and threonine present at the N-terminus.17 Other groups achieved the simplification of the complex mixture by selective capture of peptides with a low-abundant amino acid such as histidine18 or with low-frequent posttraslational modifications such as N-glycosylation.19-22 The methods developed in our laboratory for the selective isolation of peptides differs from those mentioned above because they are based on the derivatization of abundant functional groups (R- and -NH2) to restrict the presence of positive charge (at acidic pH) only to those peptides containing arginine and histidine, allowing an easier separation of neutral (R + H ) 0) and charged (R + H g 1) species by cationexchange chromatography. This principle was applied to proteome analysis in a method known as SCAPE (Selective 10.1021/pr060003w CCC: $33.50

 2006 American Chemical Society

Selective Isolation of RRnK Peptides in Proteomics

CApture of PEptides) that allows the selective isolation of either Lys-C or tryptic peptides that do not contain arginine or histidine.23 In silico analysis demonstrated that a considerable simplification of the complex mixture of peptides (4 peptides/ protein) as well as a proteome coverage of approximately 90% are achieved, as the ICAT9;10 method in a similar extent. A second approach, based on a similar principle but using a different chromatography system, has also been developed in our group (submitted for publication) to achieve 2DE-free quantitative proteomics based on the selective isolation of multiply charged peptides. Very recently, Foerttinger et al. reported a method for the selective capture of arginine-containing peptides by using selective tagging and boronate affinity chromatography.24,25 The selective isolation of peptides with free amino groups and arginine at the C-terminus is very attractive because they ionize efficiently and fragment very well in Collision Induced Dissociation (CID) experiments to yield a reliable identification in sequence databases. This method, when combined with the SILAC on 13C6-Arg, would guarantee the labeling of all selectively isolated peptides. However, it has two main limitations: first, it requires further optimization to avoid the nonspecific isolation of lysine-containing peptides, and second, the selective isolation of peptides with an abundant amino acid such as arginine causes an insufficient simplification (only 50%) of the complex mixture of peptides generated in proteome studies. In this paper, we propose a new method for the selective isolation of lysine-free peptides which are originated by the tryptic cleavage at the arginine residues (R-VX1-X2-X3-X4.... Xn-RV-; arrows and Xn indicate the cleavage site and amino acids, respectively). This method is based on the combination of tryptic digestion of biotinylated Lys-C peptides and affinity chromatography; it decreases the complexity of the peptide mixture (5 peptides/protein) considerably and, at the same time, it enables the analysis of approximately 85% of all proteomes. These peptides will be named “RRnK” because they are delimited by arginine residues (RR) in the protein sequences and do not contain lysine residues (nK). The RRnK peptides are isolated in the nonretained fraction of the affinity chromatography step and their good recoveries are advantageous in biological experiments where the amount of sample is a limiting factor. The high selectivity of the method suggests that the database search could be restricted to only RRnK peptides, and minimizes the false-positive identifications. This method is very simple, easy to implement in any protein chemistry laboratory and if used in combination with 18O-labeling or SILAC it could become a useful tool for 2DE-free quantitative proteomics.

Experimental Section Materials. Lysyl-endopeptidase (Lys-C) was from Wako, (Osaka, Japan). The 18O-labeled water (97% isotopic purity) was obtained from Euroiso-top (Sur-Yvette, France). The streptavidine sepharose high performance affinity column was obtained from GE Healthcare (NJ). The recombinant proteins, streptokinase (rSK) and R-interferon (R-IFN), were manufactured at the Center for Genetic Engineering and Biotechnology (Havana, Cuba). The biotin 3-sulfo-N-hydroxy-succinimide ester sodium salt, other reagents, HPLC solvents as well as the standard proteins: bovine seroalbumin (BSA), lysozyme C, ovalbumin (OVA), human transferrin, were purchased from SIGMA (MO).

research articles Reduction and S-Alkylation. Twenty micrograms of the artificial mixture of six proteins (rSK, BSA, R-IFN, lysozyme C, OVA and human transferrin) dissolved in 50 µL of 500 mM HEPES buffer (pH 8.5) containing 10 mM EDTA and 2M guanidium hydrochloride were reduced by adding DTT to a final concentration of 10 mM, flushed with nitrogen, and incubated 4 h at 37 °C. The free thiol groups were alkylated by adding acrylamide to a final concentration of 20 mM and the reaction proceeded for an additional 30 min at room temperature. Lys-C and Tryptic Digestions. The above protein mixture, dissolved in the same reduction and S-alkylation buffer was digested with Lys-C using an enzyme:substrate ratio of 1:200 at 37 °C during 4 h. Biotinylated Lys-C peptides were diluted 4-fold to reach a concentration of the chaotropic agent compatible with trypsin activity and the proteolytic digestion proceeded during 4 h at 37 °C using an enzyme: substrate ratio of 1:50. Complete Biotinylation of Lys-C Peptides. The Lys-C peptides were completely blocked by adding biotin 3-sulfo-Nhydroxy-succinimide ester sodium salt dissolved in water using a molar ratio reagent:amino groups of 10:1. The reaction mixture was shortly vortexed after adding the blocking reagent and returned to the ice bath (4 °C). This procedure was repeated three times at 30 min intervals. The reaction proceeded for additional 30 min at room temperature after the final addition. Removal of the O-Acylation. Drops of triethylamine were added to reach a pH of 11 and the reaction mixture was incubated for 2 h at 37 °C, and finally it was concentrated under vacuum for 30 min to remove a considerable portion of the tertiary amine before tryptic digestion. Desalting of Peptides. Before mixing the peptides derived from the compared conditions all proteolytic activity was inhibited by adding the Complete protease inhibitor cocktail (Roche Mannheim, Ingelheim, Germany) following the instructions of the manufacturer. The pool of labeled and nonlabeled peptides was desalted by using a minicolumn (2 mm × 5 cm) packed with rp-C4 from VYDAC (CA). The column was equilibrated in solution A (0.1% v/v, TFA /H2O) and the peptides were eluted by increasing the content of buffer B (0.05% v/v, TFA/CH3CN) from 5 to 80% in 10 min. The absorbance was monitored at 226 nm and the eluted peptides were concentrated to 1/3 of the total volume and dissolved in 125 mM HEPES buffer (pH 8.5) before the affinity chromatography step. Affinity Chromatography. The required volume of affinity chromatography matrix was determined by estimating of the amount of amino groups generated during the Lys-C digestion and the capacity of the matrix provided by the manufacturer (at least 300 nmol/mL). The matrix was equilibrated with 10 column volumes of 125 mM HEPES, pH 8.5 using a flow of 500 cm/h. The peptide mixture dissolved in the same equilibration buffer, but containing 2 M guanidinum hydrochloride and the complete protease inhibitor cocktail was loaded using a flow of 100 cm/h. The absorbance was monitored at 226 nm and the nonretained fraction was collected for mass spectrometric analysis. Recoveries of the Method. Equal amounts of two mixtures containing six reduced and S-alkylated proteins (rSK, human transferrin, ovalbumin, BSA, lysozyme C and alpha interferon) were separately dissolved in 500 mM HEPES (pH 8.5) containing 2 M guanidium hydrochloride prepared with H218O and H216O. The protein mixture dissolved in 18O-labeled water was Journal of Proteome Research • Vol. 5, No. 5, 2006 1205

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digested with Lys-C/trypsin as described previously, and frozen until analysis. The other protein mixture dissolved in normal water was treated following all the steps of the proposed method. The expanded regions of the mass spectrum containing the isotopic distributions of the RRnK peptides were exported as a TXT file and the relative proportions of all components (16O- and 18O1- and 18O2-labeled species) were determined by using the Isotopica software.26;27 The sum of the intensities of the 18O1- and 18O2-labeled peptides was considered as a 100% recovery of the method. The recovery was calculated by using the following formula: recovery ) [(16O)/(18O1 +18O2)] * 100%. Mass Spectrometry. Mass spectrometric measurements were performed using a hybrid quadrupole orthogonal acceleration tandem mass spectrometer QTof-2 from Waters (MA). Capillary voltages of 900 and 3000 V were used for nanoESI and LCMS/MS experiments, respectively. The cone voltage was 35 V in all measurements. The mass spectra were acquired in m/z range from 400 to 2000 Th. LC-MS/MS and Database Analysis. For these experiments, the mass spectrometer was connected on-line with an AKTA Basic liquid chromatograph (GE Healthcare, NJ) by using an RP-C18, 300 µm ID × 5 cm column (Dionex, CA). Peptides were eluted from the column using a linear gradient of acetonitrile with 0.2% formic acid from 5 to 60% for 50 min, at a flow rate of 3 µL/min. To acquire the LC-MS/MS spectra the first quadrupole was used to select the precursor ion within a window of 4 Th. A pressure of ∼3 × 10-2 Pa collision gas (argon) was used in the hexapole collision cell to yield the fragment ions. The doubly- and triply charged precursor ions to be fragmented were selected automatically once their intensity rose above a defined threshold (8 conts s-1). The instrument reverted to MS mode once the total ion count decreased below 2 count sec-1 or when the MS/MS mode had been maintained for 4 s. Data acquisition and processing were performed using a MassLynx system (version 3.5) from Waters (MA). Protein identifications were based on manual interpretation of the MS/ MS spectra. Computer programs. The program Selestact (CIGB, Havana, Cuba) coded in C for console use was developed to scan the Swiss-Prot sequence database, and calculates the number of proteins of a given organism that might be identified using the method proposed in the present work as well as for other methods previously reported in the literature,9,10,14-22 the average number of peptides per protein that could be selectively isolated. The counting was constrained to those peptides of mass comprised between 800 and 3500 Da, and the program excluded from the calculation all the N- and C-terminal peptides for reasons that will be discussed below. Two independent databases containing human receptors and kinases were constructed after filtering the swissprot database according to the content of the organism and description fields. The Isotopica software (CIGB, Havana, Cuba) is a web application suitable for quantification of labeled and nonlabeled compounds present in a mixture.26;27 It is a.NET application, coded mainly in C++, C#, and ASP.NET. This program was employed to calculate the relative abundances of nonlabeled, 18O1- and 18O2-labeled peptides in the overlapped isotopic ion distributions. The software is available through the Internet (http://bioinformatica.cigb.edu.cu/isotopica or http:// coco.protein.osaka-u.ac.jp/isotopica). 1206

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Figure 1. General strategy for the selective isolation of RRnK peptides in quantitative proteomics. The rhombuses correspond to biotinyl residues linked to primary amino groups of Lys-C peptides. RCM: reduction and carbamido methylation.

Results and Discussion Selective Isolation and Relative Quantification of RRnK Peptides. The schematic representation of the method is displayed in Figure 1. Equal amounts of proteins derived from two compared conditions are reduced, S-alkylated, and digested with Lys-C in the presence of buffers prepared with normal and 18O-labeled water, respectively. The alpha- and epsilon-amino groups of the resultant Lys-C peptides are biotinylated. A treatment with triethylamine is necessary for quenching the blocking reaction and for the complete removal of the O-acylation in tyrosine residues formed as a side product in the blocking step. The biotinylated peptides are diluted and digested with trypsin and equal volumes of the proteolytic digests derived from both conditions are mixed and desalted to discard the hydrolysis products of the biotinylation reagent which may interfere with the streptavidin affinity chromatography. All peptides containing biotinylated residues are retained onto the column while the free-lysine peptides originated by the cleavage of trypsin at the arginine residues are selectively isolated in the nonretained fraction and directly analyzed by LC-MS/MS. These peptides hereinafter will be named as RRnK. Finally, after sequence database searching and protein identification, the relative content of 16O/18O in the individual peptides is determined by using the Isotopica software26;27 in order to quantify their differential expression. In Silico Analysis of the Proposed Method. In silico analysis of different proteomes revealed that the proposed method (Figure 1), selectively isolates approximately 4 RRnK peptides per protein and at the same time an average of 85% of the whole proteome can be analyzed (Table 1). These values are

research articles

Selective Isolation of RRnK Peptides in Proteomics Table 1. Proteome Coverage and Simplification of the Complex Mixture of Peptides Achieved by the ICAT9,10 and the Present Method

proteome

total no. of proteins a

total no. of tryptic peptides/ protein b

selectively isolated peptides / proteinc (RRnK/ICAT)

proteome coveraged (RRnK/ICAT)

N. meningitidis V. cholerae M. tuberculosis E. coli S. cerevissiae A. thaliana M. musculus H. sapiens Average

1967 3785 3875 4322 4818 26003 28959 47531 -

14 15 15 15 24 14 14 22 18

4/2 4/2 8/2 5/3 4/4 4/5 5/6 6/6 5/4

77.5/75.7 80.0/72.5 94.9/72.2 85.1/76.6 82.8/86.3 86.4/90.4 86.3/91.8 87.5/91.9 84.8/82.9

a Corresponds to the total number of proteins reported in the Swiss-Prot database. b Average of the total number of tryptic peptides per protein in the analyzed proteomes considering complete cleavage of trypsin except for the K-P and R-P peptide bonds. These values are expressed as integer numbers. c In comparison with the figures shown in (b) it represents the simplification of the complex mixture of peptides and it is an average of the total # of tryptic peptides per protein that can be selectively isolated by the present method and ICAT.9;10 For both methods, only tryptic peptides with molecular masses comprised between 800 and 3500 Da were considered. These values are expressed as integer numbers. d The proteome coverage represents the % of the total number of proteins that can be identified by selectively isolating at least one RRnK peptide or a peptide containing a cysteine (in the case of ICAT 9;10) having a molecular mass within the abovementioned range (see c).

comparable to those obtained by the well-established ICAT method,9;10 developed for the selective isolation of cysteinecontaining peptides, and in consequence, they suggest the applicability of the method for proteome analysis. In this in silico analysis, the RRnK peptides detectable by the mass spectrometric analysis were those with molecular masses comprised between 800 and 3500 Da. The lower-mass limit was fixed because data acquisition started at m/z 400 and RRnK peptides are unmodified species, with two or more protonation sites (free amino group and an arginine at the C-terminus) and they are mostly detected as multiply charged ions in ESI-MS. A doubly charged peptide detected at m/z 400 accounts for a molecular mass of 800 Da, approximately. Shorter peptides (mass < 800 Da) are often difficult to analyze either because they are not retained in the rp-HPLC columns or ZipTips, or because the intensities of their corresponding singly charged ions display low intensities in the ESI-MS spectra of complex mixture of unblocked peptides and therefore, they are not automatically selected for MS/MS analysis. Also an upper-mass limit at 3500 Da was defined for calculations, because very large peptides are often not detected, the analysis of higher than triply charged peptides is generally not feasible, and their MS/MS spectra contain little information for protein identification. The proteome coverage and peptides/protein of the present method were calculated for the study of different proteomes and the results are compared with others published methods9,10,14-22 (see Supporting Information http://www. cigb.edu.cu/table Supporting Information_rrnk.pdf). SCAPE 23 and the method that isolates the histidine-containing peptides18 have the greatest proteome coverage of approximately 91%, although the selectivity of the latter method is not very high. COFRADIC,14 the present method and ICAT8,9 have similar proteome coverage from 88 to 83%. Other methods have lower proteome coverage and particularly the selective isolation of N-glycopeptides19-22 has the lowest values (56%). Regarding the

simplification of the complex mixture of peptides (peptides/ protein) most of the methods selectively isolate an average of 3-5 peptides/protein except the method that isolates N-glycopeptides19-22 which introduces the greatest simplification by the selection of 1 peptide/protein. Particularly, the method for the selective isolation of N-glycopeptides has been probed to be successful in the study the subproteomes of glycosylated proteins (such as membrane proteins), but the data shown here were obtained for all proteins reported in sequence database regardless the presence of attached N-linked sugar chains. This in silico analysis could be helpful to select the most appropriated method for studying the proteome of interest. To know whether the method could be also applicable to the analysis of low-abundant proteins with very important functions such as human kinases and receptors, two independent databases containing these classes were constructed and analyzed by the Selestact software. The results demonstrated that RRnK peptides are very well represented within the receptors (6 peptides/protein) and kinases (7 peptides/protein) and they permit coverage of these subproteomes by 90.0% and 95.8%, respectively. Application of the Method to a Standard Protein (rSK). Recombinant streptokinase (rSK) was selected as a model protein to evaluate the selectivity of the proposed method (Figure 1) because its tandem digestion (Lys-C/trypsin) generates more than 40 proteolytic peptides and it has only three RRnK peptides [221DSSIVTHDNDIFR233 (M ) 1518.71 Da), 390YTEEER395 (M ) 825.35 Da) and 396EVYSYLR402 (M ) 928.46)] with molecular masses from 800 to 3500 Da. The deconvoluted ESI-MS spectrum of the Lys-C digest of rSK is shown in Figure 2A. After the biotinylation reaction (see Experimental Section) two biotinyl residues were added to most peptides, as expected: one to the N-terminus and one to the epsilon amino group of the C-terminal lysine residues. Moreover, there are some tyrosine-containing peptides such as those labeled with arrows in Figure 2A that incorporated additionally biotinyl group (dark rhombus in Figure 2B). A similar phenomenon has been reported by Zaptacosca and Annan,28;29 who noticed that O-acylation at certain tyrosine residues seems to be inevitable during the quantitative blocking of amino groups. They also reported that attempts to limit the extent of Oacylation were unsuccessful with the primary outcome being an incomplete reaction at the N-terminus. Instead, they chose to drive the blocking of amino groups for completeness and then perform a basic treatment to de-O-acylate the tyrosine residues. A treatment of the blocked peptides at basic pH with a tertiary amine quantitatively de-O-acylates the tyrosine residues and ensures that all Lys-C peptides have the expected number of biotinyl residues (Figure 2C). The deconvoluted ESI-MS spectrum of the biotinylated Lys-C peptides digested with trypsin is shown in Figure 2D. It can be noticed that signals corresponding to certain biotinylated peptides that contain internal arginine residues (Figure 2C) were not detected after tryptic digestion (Figure 2D). The signals corresponding to blocked peptides (see empty circles in Figure 2C) that contain within their sequences the RRnK peptides of rSK are also included in this group. The signals observed in Figure 2D correspond to peptides with biotinyl residue(s) attached to the amino group(s) and unmodified peptides with masses that agree very well with those expected for nonmodified tryptic peptides of SKr (RRnK peptides, labeled with solid circles in Figure 2D). After affinity chromatography the biotinylated Journal of Proteome Research • Vol. 5, No. 5, 2006 1207

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Figure 2. Deconvoluted ESI-MS spectra of (A) Lys-C digest of rSK; (B) biotinylated Lys-C peptides; (C) biotinylated Lys-C peptides after de-O-acylation of tyrosine residues; (D) tryptic digest of the peptides shown in (C). (E) Deconvoluted ESI-MS spectra of the selectively isolated RRnK peptides of rSK by using streptavidine affinity chromatography. Signals labeled with an arrow in (A) correspond to some of the Lys-C peptides which were additionally O-biotinylated at their tyrosine residues. In (B) empty and solid rhombuses indicate the biotinyl residues added to the amino groups (either alpha or epsilon) and to the hydroxyl group of tyrosine, respectively. In (C) signals labeled with empty circles correspond to biotinylated Lys-C peptides that contain the RRnK peptides of rSK selectively isolated in (E) by affinity chromatography. Signals labeled with filled circles in (D) correspond to RRnK peptides. In (E), the calculated m/z values for the RRnK peptides are indicated in parentheses. The signal labeled with an asterisk in (E) corresponds to an ion source fragmentation ion (y”11) of the peptide 390YTEEEREVYSYLR402.

peptides were retained in the column while the second group, composed only of the RRnK peptides from rSK, was selectively isolated in the nonretained fraction (Figure 2E). The decrease in complexity of the peptide mixture achieved by the proposed method (Figure 1) can clearly be noticed by comparing Figure 2A and E. Although three major signals were detected in the nonretained fraction (m/z 1423.70, 1518.73, and 1736.84, Figure 2E), only one of them agreed with the expected molecular mass value for one RRnK peptide (221DSSIVTHDNDIFR233, M ) 1518.71 Da). 1208

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The signal at m/z 1736.80 was assigned to an RRnK peptide (390YTEEEREVYSYLR402) that contains a missed cleavage site at R395 that links the peptides 390YTEEER395 and 396EVYSYLR,402 the two expected RRnK peptides taking into account a complete cleavage of trypsin. The signal at m/z 1423.70 was also assigned to a RRnK peptide with a missed cleavage site (in R325) (321NLDFRDLYDPR331) that contains two other short RRnK peptides (321NLDFR325 and 326DLYDPR331) that originally were not considered as candidates because their molecular masses as individual species are below the previously defined mass

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Selective Isolation of RRnK Peptides in Proteomics

Table 2. Quantification Results and Recoveries of the RRnK Peptides Isolated from Six Standard Proteins in the LC-MS/MS Experiment

a

proteins

rSK (3/3)

390YTEEEREVYSYLR402 211DSSIVTHDNDIFR228

Ovalbumin (2/2)

127GGLEPINFQTAADQAR142 143ELINSWVESQTNGIIR158

Lysozyme C (2/3)

62WWCNDGRTPGSR73 46NTDGSTDYGILQINSR51

Transferrin (1/2) BSA (1/2) R-IFN (2/2)

m/z exp. (expect.)

Z

recoveriesc (%)

579.60 (579.61) 759.87 (759.86) 844.39 (844.42) 929.96 (929.99) 753.40 (753.34) 877.39 (877.42) 1093.07 (1093.06) 580.28 (580.29) 538.78 (538.80) 741.38 (741.38)

3 2 2 2 2 2 2 3 2 2

88.3 96.5 94.5 74.5 90.0 92.8 91.2 58.8 28.2 78.9

sequence of RRnK peptides

144SAGWNIPIGLLYCDLPEPR162 445MPCTEDYLSLILNR458 14TLMLLAQMR22 b) 150SFSLSTNLQESLR162 b)

quantificationd expected

exp.

75:25

75.0:24.9 74.4:25.6 82.7:17.3 84.7:15.3 26.4:73.5 21.5:78.5 67.0:33.0 57.3:42.7 31.5:68.5 32.6:67.4

83:16 25:75 66:33 50:50 33:66

a In parentheses: (detected RRnK peptides/expected number of RRnK peptides according to the amino acid sequence of the analyzed proteins). b The amino acids highlighted in bold and italics correspond to the methionine sulfoxide residues. c The recoveries were calculated by considering that the total content of 18O-labeled peptides (18O1 + 18O2) represent 100% of the recovery of the method for the selective isolation of the RRnK peptides. The formula used to calculate the recoveries is shown in the Experimental Section. The relative content of 16O/18O was determined by using the Isotopica software.26,27 d The relative quantification of the RRnK peptides was performed by calculating the ratio of 16O/(18O1 + 18O2) at the C-terminus of the peptides by using the Isotopica software.26,27

threshold (