Article pubs.acs.org/ac
Two-Dimensional Peptide Separation Improving Sensitivity of Selected Reaction Monitoring-Based Quantitative Proteomics in Mouse Liver Tissue: Comparing Off-Gel Electrophoresis and Strong Cation Exchange Chromatography Alexander Schaf̈ er,† Christine von Toerne,† Silke Becker,† Hakan Sarioglu,† Susanne Neschen,‡ Melanie Kahle,‡ Stefanie M. Hauck,*,† and Marius Ueffing†,§ †
Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany ‡ Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany § Centre of Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Tübingen, Germany S Supporting Information *
ABSTRACT: Protein expression analysis is one of the most powerful tools to further the understanding of biological systems. Progress in the field of mass spectrometry has shifted focus from gel-based approaches to the upcoming LC-selected reaction monitoring (SRM) technique which combines high technical accuracy with absolute quantification of proteins and the capability for high-throughput analyses. Due to these properties, LC-SRM has the potential to become the foundation for biomarker analysis, targeted hypothesis driven proteomic studies and contribute to the field of systems biology. While the performance of LC-SRM applied to samples from various bodily fluids, particularly plasma, and microorganisms has been extensively investigated, there is only little experience with its application to animal tissue samples. Here, we show that a conventional one-dimensional LC-SRM workflow applied to mouse liver tissue suffers from a shortcoming in terms of sensitivity for lower abundance proteins. This problem could be solved through the extension of the standard workflow by an additional dimension of separation at the peptide level prior to online LC-SRM. For this purpose, we used off-gel electrophoresis (OGE) which is also shown to outperform strong cation exchange (SCX) in terms of resolution, gain of signal intensity, and predictability of separation. The extension of the SRM workflow by a high resolving peptide separation technique is an ideal combination as it allows the addition of stable isotope standards directly after trytic digestion and will increase the dynamic range of protein abundances amenable by SRM in animal tissue.
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with enhanced sensitivity. This principle allows one to decrease the amount of time necessary for fractionation and online separation while maintaining a more consistent analysis in terms of which peptides and proteins are quantified as compared to nontargeted strategies across multiple biological samples.3 These properties have made SRM assays especially interesting for the analysis of candidate proteins in larger cohorts of biological subjects which is currently challenging when using nontargeted analyses that rely on moderate to extensive fractionation and long gradient schedules to access proteins of interest.4 Due to its excellent quantitative properties, SRM-based2 assays have become an attractive alternative to antibody-based methods for targeted protein analysis. Moreover, isoform specificity5 and
elected reaction monitoring (SRM) has emerged as the method of choice for MS-based targeted proteomics in recent years. In principal, any protein in any sample can be detected selectively and quantified accurately by measuring precursor to fragment transitions of its proteotypic peptides (PTPs).1 When combined with stable isotope labeled standard (SIS) peptides, quantification based on this approach has low technical variability and high dynamic range and is absolute rather than relative.2 As a consequence, SRM-based proteomics has become indispensable not only for biomarker studies in clinical cohorts but also for modeling approaches in systems biology. In contrast to nontargeted discovery driven proteomics, analyses have to be focused on preselected proteins based on their relevance for specific biological questions. Moreover, analyses can be restricted to a subset of PTPs that serve as a proxy for the group of proteins of interest1 and can be monitored © 2012 American Chemical Society
Received: August 10, 2012 Accepted: September 20, 2012 Published: September 20, 2012 8853
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multiplexing6 is more easily achieved using SRM than using antibody-based techniques. However, SRM assays on the current technical platforms are inferior to antibody-based methods in terms of sensitivity.7 In the present work, we sought to employ the SRM strategy in a hypothesis driven rather than discovery study of type 2 diabetes (T2D) mouse models. Candidate proteins were selected on the basis of their putative roles in T2D and lipid metabolism in the liver, because of its central role in metabolism and the importance of insulin resistance in early stages of T2D pathogenesis.8 We selected interesting candidate proteins from the large body of genetic evidence from genome wide association studies as well as the literature (Supporting Information Table S-1). Since these candidate proteins cover large dynamic ranges of expected expression levels in tissues, we found that SRM assays on nonfractionated mouse liver samples did not meet the requirements necessary for robust and successful quantification (3 PTPs with 3 transitions at signal/noise ≥ 10). Fractionation and enrichment of specific peptides has been shown to be an effective way to increase sensitivity of SRM assays in plasma. The combination of affinity chromatography (stable isotope standards and capture by antipeptide antibodies, SISCAPA)9 or strong cation exchange (SCX)10−12 with SRM is well established. Offgel electrophoresis (OGE) has been used in targeted proteomics for assay development in studies of bacteria4,13 and human tissue samples14 as well as in expression analysis of mammalian cell lines.15 Increased SRM signal intensity through OGE applied to yeast samples has been shown,16 and increased sensitivity of prostate specific antigen (PSA) quantification in plasma through the use of narrow range isoelectric focusing (IEF)17 has been described. Here, we applied fractionation of liver samples at the peptide level by either OGE or strong cation exchange chromatography (SCX) prior to SRM analyses. We show that broad isoelectric point (pI) range fractionation by OGE is more efficient than SCX in increasing the signal intensity as well as the signal-to-noise (S/N) ratios of all targeted peptides from liver tissue. However, both techniques had a highly beneficial effect on the data quality of peptide quantifications, while effort for fractionation is minimal. As a consequence, the sensitivity of our multiplexed SRM assays could be improved by a factor of approximately eight and twelve in terms of gain of signal intensity for SCX and OGE, respectively, through the use of peptide fractionation. To our knowledge, this is the first optimized workflow combining peptide-fractionation with SRM-based proteomics in an animal tissue. The presented analytical workflow is ideally suited for SRM assays as it allows the addition of SIS peptides before the fractionation step. Advantageously, technical variability of the quantitative results is not increased in comparison to standard workflows18 while data quality and sensitivity are enhanced.
24 cm IPG strips pH 4−7 were obtained from Agilent. 7 cm IPG strips pH 3−10 and Tris were obtained from GE Healthcare. Protease inhibitors (Complete with or without ethylenediaminetetraacetic acid (EDTA)) and NP40 were purchased from Roche. Light amino acids were purchased from Roth, ultrapure urea from Biomol, and trifluoroacetic acid (TFA) from Applied Biosystems. [13C615N4]-L-arginine and [13C615N2]-L-lysine with an isotopic purity of 98 atom % were obtained from Silantes. The light absolute quantification (AQUA) peptide FAILTEK was synthesized by Thermo Scientific at the highest level of purity offered (peptide purity >97%, isotopic purity >99%). Mouse Liver Samples. C3HeB/FeJ female mice were bred in house and housed in isolated ventilated cages (IVC-Racks, BioZone) supplied with filtered air, in a 12/12 h light/dark cycle (lights on from 6 am until 6 pm). Mice had free access to food and water. At 8 weeks of age, mice were killed and organs were perfused with 10 mL of ice-cold 0.9% NaCl via heart. Liver tissue samples were snap-frozen in liquid nitrogen and stored at −80 °C. All procedures for animal handling and experiments were performed in accordance with protocols approved by the Regierung von Oberbayern (District Government of Upper Bavaria). In vivo experiments were conducted in the German Mouse Clinic phenotyping platform (GMC) at the Helmholtz Center Munich. Protein Extraction and Quantification. Frozen liver samples were ground to powder using mortar and pestle under liquid nitrogen and stored at −80 °C until use. Powdered liver samples were mixed with extraction buffer (RIPA, 50 mM, Tris 150 mM NaCl, 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.5% (w/v) deoxycholate, 1% (v/v) NP40, 1× protease inhibitors) in CKM homogenization tubes (Precellys lysing kit, Bertin Technologies) on ice. Proteins were extracted by bead-based mechanical homogenization in a Precellys 24 Homogenizer (Bertin Technologies) prechilled to 0 °C by 25 s cycles of grinding at 5500 rpm. Homogenates were cleared by centrifugation at 16 000g for 30 min at 4 °C, and protein concentration was determined by BCA assay (Pierce) according to the manufacturer’s instructions. In Solution Digestion (Trypsin). For mouse liver extracts, acid labile detergent rapigest (Waters) was added to a final concentration of 2% (w/v) and protein samples were denatured and reduced using DTT at a final concentration of 3.5 mM at 60 °C for 10 min. Cysteines were alkylated using IAA at a final concentration of 11.25 mM for 30 min at RT in the dark. Subsequently, samples were diluted 10-fold with 50 mM Tris pH 8.5 and digested with trypsin at 1:20 enzyme/protein for 18 h at 37 °C. Afterward, rapigest was hydrolyzed by lowering the pH below 2 for 30 min on ice. As indicated in the results, in some instances, peptides were further purified using PepClean C18 spin column (Pierce) according to manufacturer’s instructions but loading no more than 10 μg of peptide on one column. Quantification Concatemer (QconCAT) Synthesis. Stable isotope labeled Q-peptides (Supporting Information Table S-2) were generated using the QconCAT approach according to ref 19 in the lysine and arginine auxothrophic E.coli strain AT713 (E.coli, F-, glnV44(AS), λ-, cysJ43, argA21, lysA22, rpsL104, malT1(λR), xyl-7, mtlA2, thi-1) (kindly provided by E.coli Genetic Stock Center, Yale). E.coli strain AT713(DE3) was generated using λ DE3 lysogenization kit (Novagen). 6xHisQconCAT was synthesized and subcloned into pET21a(+) by PolyQuant GmbH (Regensburg). Bacteria were transformed with pET21a-6xHisQconCAT and cultured in M9 minimal medium (4 g/L glucose, 8.5 mM NaCl, 22 mM KH2PO4, 48 mM
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MATERIAL AND METHODS Reagents and Chemicals. High performance liquid chromatography (HPLC) grade H2O, KH2PO4, N2HPO4, dithiothreitol (DTT), iodoacetamide (IAA), CaCl2, D-glucose, triton X-100, methanol, and chloroform were purchased from Merck. Sequencing grade modified trypsin, NaCl, formic acid, imidazole, MgSO4, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were obtained from Sigma-Aldrich. HPLC grade acetonitrile and sodium cholate were purchased from AppliChem. Ampholytes pH 3−10 and thiourea were obtained from Fluka. Ampholytes pH 4−7 and 8854
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Na2HPO4, 1 mM MgSO4, 0.3 mM CaCl2, 0.1 g/L [13C615N4]-Larginine, [13C615N2]-L-lysine, 0.1 g/L 18 other amino acids). QconCAT expression in 100 mL of culture was induced after bacteria had reached an OD600 of approximately 0.6 by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 2 mM for 4 h at 37 °C under vigorous shaking. Induced bacteria were harvested by centrifugation at 4000g for 10 min and subjected to two freeze thaw cycles. Lysis was achieved by lysozyme treatment (1 μg/μL lysozyme in 15 mM NaCl, 50 mM Tris, 1 mM EDTA, 0.1% Triton X-100, pH 7.4) for 30 min on ice followed by addition of denaturing Ni-NTA lysis buffer (8 M urea, 300 mM NaCl, 14 mM KH2PO4, 86 mM Na2HPO4, 50 mM imidazole, 1%(v/v) Triton X-100, 0.5%(w/v) CHAPS, 0.2%(w/v) SDS, Complete Protease Inhibitors w/o EDTA, pH 8.5) on ice. 6xHis-QconCAT was purified in two steps using immobilized metal affinity chromatography (IMAC) and OGE. First, 20 mL of crude lysate was incubated with 2.5 mL of Ni-NTA agarose (Qiagen) for 3 h at 4 °C. Subsequently, the resin was washed four times with 4 mL of the same buffer used for lysis, and the tagged protein was eluted eight times with 0.5 mL of the same buffer used for lysis without detergents supplemented with 500 mM imidazole and pH lowered to 4. Eluted 6xHis-QconCAT was precipitated with methanol/chloroform, resuspended in off-gel protein sample buffer (7 M urea, 2 M thiourea, 60 mM DTT, 1%(w/v) CHAPS, 0.4% ampholytes pH 4−7), and separated on a 24 cm linear IPG strip pH 4−7 (Agilent) using an Agilent 3100 OFFGEL Fractionator (Agilent, G3100A) into 24 fractions according to the manufacturer's instructions. Isoelectric focusing was conducted with the settings: 5000 V, 50 μA, 250 mW, and 15 °C until 50 kVh was reached. Purified QconCAT was retrieved from two fractions corresponding to pI 5.1−5.2 and precipitated using methanol/chloroform. Purity was assessed by SDS-PAGE followed by colloidal coomassie staining (RotiBlue, Roth) according to the manufacturer's instructions. Purified QconCAT was resuspended in 50 mM Tris pH 8.5, 0.2%(w/v) Rapigest to a final concentration of 1pmol/μL protein. Trypsin was added at 1:2 enyzme/protein, and the QconCAT was digested for 8 h at 37 °C. Rapigest was hydrolyzed as described for liver extracts. Aliquots of Q-Peptides were stored at −80 °C until use. Incorporation rate was assessed by measurement of the resulting Q-peptides with the final SRM method (90 min gradient). Absolute concentration of these aliquots was determined by the AQUA approach according to ref 2 using the light synthetic peptide FAILTEK. Off-Gel Electrophoresis (OGE) of Peptides. OGE was performed on an Agilent 3100 OFFGEL Fractionator with an optimized protocol using consumables from Agilent: IPG strips (pH 3−10, linear, 7 cm, GE Healthcare) were swollen in 30 mL of H2O overnight at RT. The next day, strips were dehydrated in 10 mL of acetonitrile for 20 min at RT. Dehydrated strips were inserted into off-gel trays, fixed with frames, which had been clipped to 7 wells, and rehydrated using 100 μL of 0.2% ampholytes pH 3−10 in H2O per well for 20 min at RT. Peptides were diluted in 525 μL of 0.2% ampholytes pH 3−10 in H2O and applied to the strip at 75 μL/well. Isoelectric focusing was conducted with the settings: 2000 V, 50 μA, 200 mW, and 12 °C until 15 kVh was reached. Fractions were collected, and wells were rinsed with 100 μL of 80% methanol, 0.5%TFA for 5 min to improve peptide retrieval. Corresponding fractions were pooled. Highly acidic and basic peptides were extracted from electrode papers using 100 μL of 50% acetonitrile (ACN), 0.1% TFA and combined with the most acidic and most basic fractions,
respectively. Organic solvents were removed by 45 min vacuum centrifugation at 35 °C, and fractions were concentrated using PepClean (Pierce) according to the manufacturer's instructions to a final volume of 20 μL. Fractions five and six, counted from the anode, were pooled. Average pH values were measured on a strip run without peptides in parallel using a micro pH electrode (Mettler Toledo). Strong Cation Exchange (SCX) Separation of Peptides. Off-line SCX fractionation of peptides was conducted on an Ettan micro-HPLC (GE Healthcare) equipped with a C18 trapcolumn (1 × 5 mm PepMap100 C18, 5 μm, 100 Å; LC Packings) and an SCX analytical column (0.3 × 150 mm PolysulfethylA SCX, 5 μm, 300 Å, PolyLC) in backflush configuration. Peptides were loaded onto the trap column in 0.1% TFA and desalted at 30 μL/min for 10 min. Transfer of peptides from C18 to SCX was done by a short organic bump using 80% acetonitrile, 0.1% formic acid in water at 3 μL/min for 3 min. Subsequently, peptides were separated using 5% acetonitrile, 0.1% formic acid in water (A) and 5% acetonitrile, 0.1% formic acid, 500 mM NaCl in water (B) at a flow rate of 5 μL/min, according to the following gradient schedule: 0−5 min, 2% B; 5−25 min, 2−30% B; 25−30 min, 30−65% B; 30−31 min, 65−95% B, hold 95% for 6 min and re-equilibrate at 2% B for 15 min. Peptide elution was monitored at 214 nm, and 5 min fractions were collected. LC-SRM/MS. LC-SRM/MS analysis was performed on a Tempo nano MDLC system (AB Sciex) coupled online to a QTrap 4000 (AB Sciex) mass spectrometer by a nano spray III ion source. Peptides were loaded onto a nano trap column (0.3 × 5 mm, PepMap100 C18, 5 μm, 100 Å; LC Packings) in 0.1% TFA at a flow rate of 20 μL/min for 5 min and separated on a C18 analytical column (0.075 × 150 mm, PepMap100 C18, 3 μm, 100 Å, LC Packings) by a gradients of 90, 120, or 180 min using 2% acetonitrile in 0.1%formic acid in water (A) and 0.1% formic acid in 98% acetonitrile (B) at a flow rate of 250 nL/min. The 90 min gradient settings were: 5−65 min, 5−40% B; 65−70 min, 40− 90% B; 70−72 min, 90% B; 72−79 min, 90−5% B, reequilibration for 10 min. The 120 min gradient settings were: 5−95 min, 5−40% B; 95−100 min, 40−90% B; 100−102 min, 90% B; 102−110 min, 90−5% B, re-equilibration for 10 min. The 180 min gradient settings were: 5−155 min, 5−35% B; 155−160 min, 35−90% B; 160−162 min, 90% B; 162−170 min, 90−5% B, re-equilibration for 10 min. Electrospray ionization was maintained with curtain gas set to 14 psi, a spray voltage of 2.6 kV, ion source gas set to 30 psi, and an interface heater temperature of 170 °C. During establishment and validation phase, peptides were detected in SRM mode with Q1 and Q3 set to unit resolution and dwell times ranging from 20 to 50 ms. SRM dependent MS/MS spectra acquired in enhanced product ion scan (EPI) mode were triggered if transitions exceeded a threshold of 200 counts. EPI scans were acquired by summing two scans at speed 4000 Da/s, Q1 set to unit, Q0 trapping enabled, and fixed fill times ranging from 300 to 800 ms. Collision energy for EPI scans was determined from a charge state specific linear curve dependent on m/z. Validated SRM assays were combined into one multiplex method in which peptide measurements were conducted at unit resolution in Q1 and Q3 using scheduled SRM with a retention time window and a target scan time adjusted gradient length according to observed peptide separation characteristics. Retention time window and target scan time were 2 min/5s for 90 min, 5.5 min/6s for 120 min, and 11.5 min/6s for 180 min gradients. Establishment and Optimization of SRM Assay for 18 Candidate Proteins. Mouse liver extracts were fractionated by 8855
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correlation cofficients of signal gain versus GRAVY values were calculated using Graphpad Prism 4.0. Gene ontology (GO) terms were retrieved using AmiGO amigo.geneontology.org/. Assessment of Limit of Quantification (LOQ). Varying concentrations of heavy Q-peptides were spiked into a constant background of mouse liver in solution digest. Final amounts on column per injection were 0.5, 2, 10, 50, and 100 fmol Q-peptides and with mouse liver peptide kept constant at 1 μg. Each concentration was measured four times using the final 90 min gradient method. S/N and area values were averaged between technical replicates, and transitions that had lower linearity than r2 = 0.95 were removed; the lowest concentration at which S/N exceeded 10 was considered as being above LOQ for the specific transition.
SDS-PAGE and subjected to in gel digestion as described in ref 20. Briefly, 60 μg of protein was separated on a 10% discontinuous SDS-PAGE gel, stained with coomassie, cut into five fractions, reduced and alkylated with DTT and IAA, and digested overnight with trypsin at an enzyme/protein ratio of approximately 1:50. Peptides were eluted using 100% ACN, 0.5% TFA, dried by vacuum centrifugation, and reconstituted in 2% ACN, 0.5% TFA. PTPs were selected on the basis of the criteria outlined in ref 21: length 5−30aa, no cysteine or methionine containing peptides, no missed cleavage sites, unique to the target protein as assessed by BLAST search (http://blast.ncbi. nlm.nih.gov) against the “all non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF” database with organism filter set to Mus musculus. Gel-fractionated samples were subjected to nontargeted proteomics analysis on an LTQOrbitrap XL (Thermo)/Ultimate 3000 (Dionex) LC-MS system, and peptides of target proteins detected in this analysis were preferentially selected from the list of proteotypic peptides. Assays were established by screening for PTPs of each protein in the fraction corresponding to its molecular weight (MW) using the 90 min SRM-MS/MS method described before (see section LC-SRM/MS). A variable number of proteotypic peptides were screened with the aim of obtaining three peptides with three transitions for each protein. Over the course of the establishment phase, up to 14 peptide per protein and 16 transitions per peptide were analyzed supported by the generation of MS/MS data during the screening runs. Supporting Information Table S-1a contains a full list of tested peptides and transitions. Peptides in the final assay were validated by manual inspection of MS/MS as well as coelution with their corresponding heavy Q-peptide. Collision energy (CE) optimization of selected transitions for the final assay was done by testing nine CE values in two volt increments centered on the initial value determined by charge state and m/z during LC-MS runs using gel-fractioned samples used for establishment. The optimum value was determined by manual inspection of LC-MS runs in the Analyst 1.5 software. Data Processing and Quantitative Analysis. Raw data acquired using Analyst 1.5 was imported into Multiquant 2.0 (AB Sciex) for quantitative analysis. Peak areas were determined by integration of SRM traces with the MQ4 algorithm using the following settings: Gaussian smooth width, 3 points; min peak width, 3 points; noise percentage, 85%; baseline subtraction window, 2 min; peak splitting, 2 points. Signal-to-noise ratios were determined using the SignalFinder algorithm with the settings: confidence threshold, 50%; no global baseline, allow nonlinear baseline. Multiquant results were exported to Excel 2007 and Graphpad Prism 4.0 for further analysis. Visual comparison of SRM traces was done with Skyline 1.2 using Savitzky−Golay smoothing of all traces. MS/MS spectra were exported with the built-in Mascot script of Analyst and searched against the Ensembl mouse database using Mascot (Matrix Science, version 2.3.02) using the following settings: precursor mass tolerance, 0.8 Da; fragment tolerance, 0.8; allowed missed cleavages, 1; fixed modifications, carbamidomethylation (C); variable modifications, oxidation (M), deamidation (N,Q); instrument, ESI-TRAP. Hierarchical clustering was done with Perseus (MPI Biochemistry, Martinsried). Theoretical pI values were calculated from peptide sequences using the approach of Bjellqvist et al22 implemented on http://web.expasy.org/compute_pi/. Grand average of of hydropathy (GRAVY) values23 were determined using http://www.gravy-calculator.de/. Spearman
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RESULTS AND DISCUSSION Development of the Multiplex SRM Assay for a Panel of Candidate Proteins. We selected proteins involved in Table 1. Proteins Quantified by the SRM Assaya
accession
protein
Q8K010 P56528
5-oxoprolinase CD38 ADP phosporibosyltransferase electron transfer flavoprotein DH elongation of very long fatty acids 2 fatty acid desaturase 1 glucokinase glucokinase regulatory protein glutaminase liver glutaryl-CoA DH long-chain fatty acid CoA ligase 1 long-chain specific acylCoA DH medium-chain specific acyl-CoA DH proline DH protein gamma glutamyltransferase 2 short-chain specific acylCoA DH solute carrier glucose GLUT2 sorbitol dehydrogense uricase
Q921G7 Q9JLJ4 Q920L1 P52792 Q91 × 44 Q571F8 Q60759 P41216 P51174 P45952 Q9WU79 P21981 Q07417 P14246 Q64442 P25688
gene name
peptides
abundance [fmol/μg total protein]
Oplah Cd38
3 3
3.0 2.1
Etfdh
4
12.0
Elovl2
1
3.7
Fads1 Gck Gckr
3 4 4
3.0 3.4 2.7
Gls2 Gcdh Acsl1
4 5 4
3.7 50.0 30.0
Acadl
4
31.4
Acadm
5
16.7
Prodh Tgm2
3 5
8.6 9.7
Acads
4
36.7
Slc2a2
4
19.6
4 4 Σ 68
103.0 230.7
Sord Uox
Proteins and their numbers of peptides targeted in the final assay are noted. Abundance was calculated as average of the abundance of quantified peptides from the fractionation data from a single mouse liver. a
carbohydrate and lipid metabolism and with known genetic association with T2D24−29 and used gel fractionated pools of mouse liver samples containing the endogenous proteins as source material for the development of our assay to be used for different T2D mouse models. PTPs were selected from shotgun proteomics data or, if target proteins were not contained in these data sets, Peptide Atlas30 was used to select peptides likely to be observed. During the course of the development phase, up to 14 8856
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Figure 1. Optimization of one-dimensional LC-SRM performance. (a) Effect of gradient length and clean up on the number of quantifiable (3 transitions, S/N ≥ 10) peptides. 500 ng of liver protein digest spiked with 300 fmol of Q-peptide was separated with the indicated gradient length with or without clean up by C18 SPE PepClean (PC). Bars correspond to four technical replicates mean ± standard deviation (SD) (b) Column loading. Specified amounts of liver protein digest spiked with 300 fmol Q-pepides were analyzed with the 90 min gradient without PC. Bars correspond to four technical replicates mean ± SD. (c) Peak widths. Average peak widths (full width at half-maximum, FWHM) of the Q-peptides from (a). Each dot corresponds to one peptide.
Figure 2. Improvements of the SRM assay by fractionation at the peptide level. (a) Number of quantifiable peptides (3 transitions, S/N ≥ 10) for direct injection (1 μg), SCX (6 fractions, 20 μg), or OGE (6 fractions, 50 μg). Bars correspond to four technical replicates mean ± SD (b) Gain of signal intensity. Sum of all transitions belonging to a peptide in SCX or OGE was averaged over four technical replicates and normalized to the direct injection data. Each dot corresponds to one peptide with median values indicated. (c) Improvement in S/N. Transition values were first averaged over technical replicates and then for all transitions of each peptide. Bars indicate median values.
labeled with lysine-8 and arginine-10 using the lysine/arginine auxotrophic E.coli strain AT71332 (Supporting Information Figure S-1b). Retention time matching between the heavy and endogenous light peptides could verify the specificity of the final assay for all peptides. In order to benchmark instrument performance and development of our assay, we performed serial dilution experiments of heavy Q-peptides in complex background (1 μg mouse liver digest) to estimate the LOQ and linearity of response for each transition. Using criteria commonly applied in literature,10,11,33 namely, using only the best transition of each peptide to characterize its LOQ, we found LOQ values of
