MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting*

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance. Molecular  Despite well-known technical limitations and numerous application pitfalls, Western blotting (WB) 1 remains one of the most widely used tools in analytical biochemistry (reviewed in (1)(2)(3)(4)(5)(6)(7)). WB conveniently provides a semi-quantitative estimate of the protein abundance directly from crude cell or tissue extracts. Quantification capabilities of WB, particularly its linear dynamic range, have been improved by using secondary antibodies bearing fluorescent labels and advanced systems for the optical readout of the abundance of recognized protein bands (8). This, however, has not alleviated the critical requirement of having antibodies with high and specific affinity toward target proteins (9).
Because of the attomole sensitivity, protein identification confidence, quantification accuracy, analyses throughput (reviewed in (29)) and, last but not least, the availability of highend mass spectrometers proteomics has had a major impact on the entire field of molecular and cell biology. However, it is often perceived as a tool for monitoring global proteome-wide perturbations that is too cumbersome and inflexible for hypothesis-driven studies encompassing a limited selection of proteins that need to be quantified in many biological conditions. High costs and technical hurdles of proteome-wide labeling of tissues or entire model organisms with stable isotopes; cumbersome preparation of clean protein extracts; inconsistent quality of synthetic peptide standards; biased quantification of membrane and modified proteins are common bottlenecks in targeted proteomics applications.
Here we report on a method we termed MS Western that provides multiplexed absolute (in moles) antibody-free quantification of dozens of user-selected proteins from unlabeled cell and tissue lysates that combines sample preparation versatility of conventional WB with the specificity, accuracy, and sensitivity of LC-MS/MS. software were used for extracting peptide features from LC-MS/MS data sets.
Experimental Design and Statistical Rationale-For benchmarking and validation of MS Western we designed four QconCAT chimeric proteins (CP01 to CP04) having MW in the range of 35 to 264 kDa and comprising proteotypic peptides from proteins of different properties (e.g. cytosolic, transmembrane), size (from 8 to 2065 kDa) and organismal origin. Knock-down experiments in HeLa cells were performed in biological triplicates and analyzed by LC-MS/MS in technical duplicates. MS Western benchmarking experiments were performed in technical duplicates for each of 15 samples made by successive dilution of a total protein extract of HeLa cells. Core histones were quantified in zebrafish embryos in three biological replicates and LC-MS/MS run were acquired in technical duplicates. Wherever applicable, we provide standard deviation (ϮS.D.), coefficient of variance (CV) and robust coefficient of variance (rCV) calculated as 1.4826 times the median absolute deviation (32).
Quantification of Proteins Spiked into an E. coli Lysate-The overnight culture (OD 600 ϭ 1.5) of E. coli strain BL21 (DE3) T1 pRARE was pelleted by centrifugation at 5000 rpm for 5 min at 4°C (JLA 8.1000 centrifuge from Beckman Coulter, Brea CA). The cell pellet was re-suspended in 2ϫ PBS, lysed in the presence of protease inhibitors and the total protein content was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equimolar mixtures containing 25 fmol to 25 pmol of each protein standard (stock concentrations were quantified by amino acid analysis) were spiked into 50 g of an E. coli protein extract and subjected to 1D SDS-PAGE as above. On electrophoresis gel slices corresponding to the apparent MWs of spiked proteins were excised, codigested with gel bands of CP01 and recovered peptides quantified by LC-MS/MS.
Absolute Quantification of Human Recombinant Histone H4 -Aliquots of a stock solution of recombinant human H4 with the concentration of 1 mg/ml were appropriately diluted and mixed with equal volumes of 2ϫ Laemmli buffer. Three samples containing 0.05, 0.1, and 0.3 g of histone H4 were subjected to 1D SDS-PAGE as described above. In parallel, the CP02 and an aliquot containing 0.066 g of Pierce BSA standard were run on a separate gel. The excised bands of histone H4, CP02, and BSA were mixed and codigested with trypsin. After in-gel codigestion and extraction, tryptic peptides were reconstituted in 46 l of 5% aqueous FA and 5 l were analyzed by LC-MS/MS.
Knock-down Experiments in HeLa Cells-AKT1, CAT, PLK1, and TUBA4A genes were knock-down (KD) in HeLa cells using RNAi (Eupheria Biotech, Dresden, Germany). KD experiments were performed in triplicate; renilla luciferase transfections as a RNAi specificity control were performed on a separate plate. Further details are provided in Supplementary Methods; details on antibodies and RNAi probes are in supplemental Tables S6 and S7, respectively.
Comparison of Protein Quantification by MS Western and LI-COR Odyssey-HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1% penicillinstreptomycin (Gibco™ Life Technologies). 1 ϫ 10 7 HeLa cells were re-suspended in 100 l RIPA buffer containing Complete Protease Inhibitor Mixture, EDTA-free (Roche). Equal volume of 2ϫ Laemmli buffer was added and incubated at 80°C for 15 min with intermittent vortexing. The supernatant was transferred to a new vial and further diluted with RIPA:2x Laemmli buffer (1:1, v/v). Samples obtained at each dilution step were subjected to 1D SDS-PAGE on two different gels. One gel was analyzed by LI-COR Odyssey and another by MS Western using bands of CP03 and BSA as standard and reference proteins, respectively. In each dilution step an equivalent amount of target proteins was digested and injected into LC-MS/MS or imaged by the Odyssey.

Monitoring the Kinetics of In-Gel Digestion of HeLa Proteins-
Protein extracts of ca. 1 ϫ 10 7 HeLa cells were prepared as described above, however in one series of experiments, the same amount of cells was homogenized in a twice larger volume of the buffer. Aliquots of cell extracts each equivalent to 4% of the total amount of recovered protein material were loaded onto multiple lanes of polyacrylamide mini-gels. On SDS-PAGE, gel slabs whose M r corresponded to TUBA and CAT proteins were excised; gel bands of CP03 and of the reference protein BSA were mixed with each gel slab and all samples were in parallel digested with trypsin. After the specified periods of time one sample per each digestion experiment was withdrawn, peptides were extracted from the entire in-gel digest and quantified by LC-MS/MS as described above. Each sample was analyzed in technical duplicates. The amount of protein digested at each time point was calculated by averaging the amounts of five independently quantified peptides from TUBA and BSA and of three peptides from CAT.
Absolute Quantification of Histones in Zebrafish Embryos by MS Western-Wild-type (TLAB) zebrafish embryos were dechorionated immediately on fertilization, synchronized and allowed to develop to the desired stage at 28°C. Ten embryos per developmental stage (except five embryos for 1-cell stage) were manually deyolked and snap frozen in liquid nitrogen. Samples were boiled in the Laemmli buffer at 98°C for 10 min and subjected to SDS-PAGE. A single gel slab containing all histones was excised from each sample lane and histones were quantified by MS Western using bands of CP02 and BSA as standard and reference proteins, respectively. 10% of the total amount of recovered tryptic peptides were injected into LC-MS/MS.
Absolute Quantification of Histones in Zebrafish Embryos by LI-COR Odyssey-Zebrafish embryos were collected at the specified developmental stages (n ϭ 5 for H3 and H2B; n ϭ 10 for H2A and H4). Embryos were processed as described above and total protein extracts were subjected to SDS-PAGE. Proteins were blotted onto a nitrocellulose membrane (GE Life Sciences). Primary antibodies (supplemental Table S8) were incubated at room temperature for 1 h or at 4°C overnight; secondary antibodies (supplemental Table S8) were incubated at room temperature for 45 min. Proteins were quantified by LI-COR Odyssey using tubulin as a loading control. Standards of recombinant human histones were used for making calibration plots for the quantification of corresponding zebrafish homologs.

RESULTS AND DISCUSSION
MS Western: Protein Quantification Concept and Workflow-Effectively, MS Western merges the three established analytical approaches: GeLC-MS/MS (30, 31, 34 -36); proteotypic peptides clubbed with "top N peptides" protein quantification (31,(37)(38)(39)(40)(41)(42) and QconCAT synthesis of isotopically labeled protein chimeras comprising concatenated sequences of proteotypic peptides (14,15,32). We termed this method as MS Western to underscore that it is targeted (rather than global), quantitative, relies on SDS-PAGE of crude protein extracts and in this way, is in line with classical Western. However, because of mass spectrometry readout, it requires no blotting and, most importantly, no antibodies.
To quantify a protein by MS Western we first selected a few (typically, three to six) proteotypic peptides (37,39) in preliminary GeLC-MS/MS experiments, which also verified the position of bands of target proteins at the electrophoresis gel lanes separating crude protein extracts. However, peptides could be also picked from collections of LC-MS/MS spectra (43,44) or predicted by software (45,46).
In the same way, we further selected proteotypic peptides from two reference proteins-in this work we used glycogen phosphorylase (GP) and bovine serum albumin (BSA). Peptide sequences from the target and reference proteins were concatenated in-silico in an arbitrary order except that peptides from the same protein were positioned successively. The entire stretch of peptide sequences was flanked at the N-and C-termini with the sequences of twin-strep-tag followed by a 3C protease cleavage site and His-tag, respectively (Fig. 1). These tags protect target peptides from exopeptidase degra- Sequences of proteotypic peptides (schematically shown as boxes) from each target protein (color-coded) are in-silico concatenated into a single chimera, flanked with peptide sequences from the reference proteins GP (at the N-terminus side) and BSA (at the C-terminus side) and with two affinity tags together with 3C cleavage site.
dation and, only if deemed necessary, could be used to enrich the expressed chimera from a whole cell lysate. Altogether we designed four project-specific chimera proteins (CP) ranging in size from 35 to 264 kDa that encoded, in total, more than 300 proteotypic peptides from 58 individual proteins (supplemental Fig. S1, S3, S5, and S7). The design rationale was the same as in QconCAT proteins (14,15) and here we use CP acronym solely for the presentation clarity. All CPs made in this work were highly expressed in E.coli (supplemental Fig.  S2, S4, S6, and S8) and incorporated more than 99.5% of heavy arginine and lysine residues (supplemental Fig. S9).
In contrast to the relative (fold change) quantification, absolute (in moles) quantification critically depends on the exactly known concentration of internal standard(s). In Qcon-CAT and related methods it is usually determined by the amino acid analysis or photo-or colorimetric assays (15). This, however, requires highly purified CPs and is prone to batch-to-batch variations and inter-laboratory inconsistency. Instead, MS Western uses a simple workaround solution that requires no CP purification (Fig. 2).
To quantify proteins of interest a cell or tissue lysate was subjected to SDS-PAGE. Gel slab(s) approximately matching the M r of target protein(s) were excised and mixed with the band of the CP excised from a lane with E. coli lysate and with the band of a reference protein (e.g. BSA). The three bands were codigested with trypsin, recovered peptides were analyzed by LC-MS/MS and quantified by considering the abundance of XIC peaks of matching pairs of light and heavy peptides. Molar abundance of each quantified protein (including CP) was calculated by averaging molar abundances of several (typically, three to six) proteotypic peptides. Gel bands of CP, BSA and the gel slab with target protein(s) were in-gel digested together, light and heavy peptides were extracted from the same digest and quantified in the same LC-MS/MS experiment. Hence, in MS Western workflow the molar amount of each target protein was inferred from the molar amount of the CP, which in turn was referenced to the known molar amount of the BSA standard. However, there was no need in purifying CPs to homogeneity or maintaining their stock solutions with the exactly known concentration. In all experiments absolute (molar) quantities of all CPs were referenced to the same commercial protein standard (Pierce BSA) with the guaranteed chemical purity, stock concentration and batch-to-batch reproducibility.
MS Western protocol should be adjusted according to the M r and abundance of target proteins. Coomassie staining was not intended to visualize bands of target proteins. Instead, proteins were excised within slightly larger (typically, Ϯ 20 kDa) gel slabs (Fig. 2C) centered at their expected M r and verified by preliminary GeLC-MS/MS. If a target protein smeares across several adjacent gel slices, molar amount (but not raw peak area) of each peptide in each slice should be summed up. For optimal quantification the abundance ratio between light and heavy peptides should be adjusted by considering the isotopic purity (Ն99.5%) of heavy arginine and lysine amino acids, rather than large (better than 10 4 -fold) linear dynamic range of Orbitrap analyzers (47). We typically codigested ca. 100 fmols of CPs, depending on the abundance of target proteins. If required, the detection sensitivity could be further enhanced by using DDA with an inclusion list of expected m/z of target peptide precursors, by t-SIM or parallel reaction monitoring (PRM) (48).
Taken together, a vastly simplified MS Western workflow offers several experimental advantages. First, both CP and target proteins are independently quantified in the same experiment by several proteotypic peptides and it is expected that they should produce quantitatively consistent determinations (31,40). Second, MS Western protocol only requires femtomole amounts of isotopically labeled standards. All four CPs were produced with high yield in E.coli (supplemental Fig.  S2A, S4A, S6A, and S8A) and ca 1 ml of the cell culture provided them in hundred picomole quantities. In our experience none of expression experiments failed or low yield of CPs required their enrichment before MS Western quantification. Also, we did not maintain prequantified CP stocks, but simply excised their bands directly from a gel loaded with E. coli lysate. Third, because proteins were solubilized in 4% SDS and analyzed by GeLC-MS/MS the quantification was not affected by the solubility or purity of CPs and target proteins. SDS-PAGE also improved both the dynamic range and sensitivity of peptide detection. In contrast to a gel-free "mass western" approach by Arnott et al. (27) that is also compatible with SDS extraction of proteins our method enables their absolute (rather than relative) quantification (also including membrane proteins) and uses no chemical derivatization and/or multistep clean-up of heavy and light pairs of proteotypic peptides. And finally, in contrast to conventional WB, MS Western does not rely on antibodies and ordering a synthetic gene at the current price level of ca 10 to 15 Eur per peptide with guaranteed quality and a few weeks delivery time is simply incomparable with costs and efforts required for generating and validation of reliable monoclonal antibodies.
Validation and Properties of MS Western-Although the concept of MS Western looked appealing, a major question remained if in-gel codigestion of relatively pure CPs, of target protein(s) embedded into a polyacrylamide gel matrix together with abundant background proteins and of pure BSA standard delivered the same yield of proteotypic peptides?
Gel bands of each of these five target proteins were codigested with the band of CP01. The extracted peptides were analyzed by LC-MS/MS and relative abundances of light (nor-malized to all light) and heavy (normalized to all heavy) proteotypic peptides were compared (supplemental Fig. S10). We observed that within 31 peptide pairs the relative abundances varied by less than 5% (supplemental Fig. S10), despite light and heavy peptides originated from structurally different chimera and endogenous proteins. Indeed, before in-gel digestion chimera, target and reference proteins were fully denatured first by SDS and then by acetic acid/methanol during Coomassie staining. Also, because of preseparation of crude extracts by SDS-PAGE, only a small fraction of a background proteome was codigested together with target protein(s). In line with previous findings (32), we observed no . The CP is quantified by comparing the abundances of XIC peaks of unlabeled (from reference protein BSA) (E) and labeled (from the CP) (F) peptides (G). Next, the amount of target protein is inferred from the ratio of abundances of XIC peaks of matching unlabeled (from the target protein) (H) and labeled (from the CP) (I) proteotypic peptides (J) and from the amount of CP determined as described above. For clarity, only two (out of several) matching pairs of labeled/unlabeled peptides are shown. A solubilized pellet of E. coli cells (panel B) was subjected to SDS-PAGE without prior enrichment of the CP. Quantification of a peptide from norpA protein from D.melanogaster using CP04 chimera is shown here as an example. noticeable impact of the size and composition of both target protein and CPs and speculated that SDS-PAGE and in-gel digestion might even relax the constraints (49) applied for the selection of proteotypic peptides.
In only a few instances (supplemental Fig. S10B and S11F) not all relative abundances matched because of trypsin miscleavages or post-translational modifications (PTMs). However, irrespectively of why they mismatched, "problematic" pairs of peptides could be spotted by their discordant relative abundances and disregarded from the protein quantification.
We next asked if the likeness of peptide relative abundances in in-gel digests of CP and of target proteins warranted their accurate molar quantification. As a test bed, we used the CP02 comprising peptides from the four core histones H2A, H2B, H3, and H4 from D.rerio (supplemental Fig.  S3, S4 and supplemental Table S2). We also obtained a standard of human recombinant histone H4 supplied as a stock solution with the exactly known concentration. Human and zebrafish histones H4 share 99% full-length sequence identity (supplemental Fig. S12) and we tested if human H4 could be quantified using identical peptides from CP02 whose molar abundance was referenced to the BSA standard. Three aliquots containing different amounts of human H4 were subjected to SDS-PAGE and quantified by MS Western. Relative abundances of light peptides from BSA and histone H4 and corresponding heavy peptides from CP02 were in a good agreement ( Fig. 3A and Fig. 3B, respectively). Relative abundances of one BSA (QTALVELLK) and one histone H4 (TVTAMDVVYALK) peptide mismatched because of trypsin miscleavage caused by flanking dibasic amino acid residues in the sequences of endogenous proteins (45, 50 -52). When the areas of XIC peaks of miscleaved peptides were added into the calculation, the expected relative abundances were restored. Next, for each peptide and each amount of histone H4 we calculated the ratio of relative abundances of their light (normalized to all light) and heavy (normalized to all heavy) peptide forms. If these normalized relative abundances remain the same then their ratio should be close to the value of 1.0, which was also consistent with our findings (Fig. 3C). Altogether, in the three independent experiments MS Western quantification relying on five proteotypic peptides (Fig. 3B) correctly determined different molar amounts of histone H4 loaded on the gel (Fig. 3D).
Finally, we checked if the target, chimera and reference proteins were in-gel codigested each at its own kinetics and if matching of their digestion rate was required for accurate absolute quantification? To this end, we monitored in-gel digestion of ␣-tubulin (TUBA, 50 kDa) and catalase (CAT, 60 kDa) in a SDS-PAGE separated total protein extract from HeLa cells. Gel slabs were excised at the correspondent M r , codigested with bands of 72 kDa chimera protein CP03 (supplemental Fig. S5, S6 and supplemental Table S3) comprising proteotypic peptides from TUBA and CAT and with bands of the reference protein BSA. Peptides extracted from in-gel digests were quantified by LC-MS/MS to produce the kinetic plots in Fig. 4A, 4B, and 4C. In the HeLa extract the abundance of TUBA and CAT differed by ca 100-fold (Fig. 4B, 4C) and they were digested together with ca 1100 comigrating background proteins. As a consistency check, we independently digested two loadings of the same extract whose total protein amount differed by 2-fold. In line with the previous report on in-solution digestion of chimera proteins (53), CP03 was digested in-gel within a few minutes. The digestion of TUBA and CAT was complete after ca 6 h, whereas MS Western protocol relies on overnight digestion. Interestingly, not all proteotypic peptides were produced at the same rate (Fig. 4D, 4E). The ratio of relative abundances of light and heavy forms of most peptides plateaued at the expected value of 1.0 already after 3 h, consistently with the kinetic plots in Fig. 4B, 4C. However, two peptides showed deviating trends. The ratio of relative abundances of peptide QTALVELLK was lower than expected (Fig. 4D, supplemental . Consistently with earlier reports on the specificity of trypsinolysis of peptides flanked with successive Arg, Lys residues (45, 50, 51, 54) the yield of QTALVELLK did not improve at extended digestion times, whereas its release from CP03 was rapid and complete. Peptide QLFHPEQLITGK from TUBA was also produced at the slow rate, however its release was complete after 12 h (Fig. 4E, supplemental Fig. S14). Importantly, biased yield of both peptides at all digestion time points was clearly reflected by deviating ratios of relative abundances of their light and heavy forms and supported the informed decision on accepting or excluding them from protein quantification.
Benchmarking Protein Quantification by MS Western-We further benchmarked MS Western quantification in three ways. First, we checked if its linear dynamic range was affected by a concomitant protein background. Second, we tested if both WB and MS Western produced quantitatively concordant determinations of endogenous proteins in whole cell lysates. Finally, we benchmarked MS Western sensitivity and dynamic range against currently the most advanced quantitative Western blotting system LI-COR Odyssey (further termed as Odyssey) that relies on near-infrared immunofluorescent readout of the abundance of antibodies recognizing target proteins.
We first analyzed a series of eleven samples containing 25 fmol to 25 pmol of an equimolar mixture of five proteins (GP, BSA, ENO, ADH and UBI) spiked into 50 g of E. coli protein extract. Each sample was subjected to SDS-PAGE and the five proteins were quantified by MS Western by codigesting their bands with the band of CP01. Closely migrating bands of ENO and ADH (⌬M r ca 10 kDa) were excised and digested together. Calibration graphs were produced by plotting the average of ratios of absolute abundances of light and heavy forms of proteotypic peptides pertinent to each protein against the injected amounts of protein digests and were linear within the entire range of protein loadings (Fig. 5A). Calibration plots for 21 individual peptides used for making Fig. 5A are provided in supplemental Fig. S15 and suggested that peptides recovery was independent of protein size and percentage of acrylamide in digested gel slabs and was not affected by protein background.
Next, we tested if WB on LI-COR Odyssey and MS Western provided concordant quantification of endogenous proteins in whole cell lysates. In contrast to MS Western, pure protein standards are required for the absolute quantification by the Odyssey because slopes of calibration plots relying on integral intensities of visualized bands differ between individual proteins. Therefore, to compare the two methods, we depleted several proteins in HeLa cells by RNAi. We then analyzed the same extracts from control and KD cells by the Odyssey and by MS Western and checked if they reported concordant changes in protein abundances.
For MS Western quantification we designed a 72 kDa chimera protein CP03 comprising 47 proteotypic peptides from six human proteins: CAT, TUBA, PLK1, AKT1, GAPDH and PTEN (supplemental Fig. S5, S6 and supplemental Table S3) for each of which commercial monoclonal antibodies are available. Both Odyssey and MS Western determined quantitatively concordant levels of endogenous TUB, PLK1, CAT,  and AKT1 in KD-cells as compared with the luciferase control (supplemental Fig. S16). Interestingly, MS Western could separately quantify the relative abundances of TUBA4A and TUBA1A sharing 96% of the full-length sequence identity. The abundance of both proteins was affected by RNAi at the same extent, however they were not distinguished by the Odyssey (supplemental Fig. S16F).
We used the same proteins to benchmark the dynamic range and sensitivity of MS Western quantification in comparison to the Odyssey. To this end, samples of successively diluted total protein extract from HeLa cells were loaded on two different gels. We adjusted the loaded volumes such that, for each dilution, the same amount of protein extract (here exemplified as an equivalent number of extracted cells) was subjected to Odyssey imaging and injected into the LC-MS/ MS. Therefore, protein molar amounts (as determined by MS Western) and integrated intensities of protein bands (determined by Odyssey) could be correlated with no further adjustment (Fig. 5B, 5C). Fig. 5D and 5E compare the dilution plots of TUBA quantified by MS Western (as TUBA1A) and by the Odyssey (as TUBA1A and TUBA4A), respectively. In WB experiments GAPDH served as a loading control and its abundance followed the dilution of HeLa extract. In MS Western an equal amount of CP03 was codigested with each sample and therefore its constant abundance evidenced the analyses consistency. Compared with the Odyssey, MS Western showed at least 60-fold better sensitivity (down to 1.5 fmol of TUBA1A) along with higher dynamic range (Ͼ16000-fold) and excellent linearity (r 2 ϭ 0.9983) (Fig. 5E). In the middle of the plot ( 5D) the Odyssey also showed good linearity toward both GAPDH and TUB1A/4A. However, at lower loadings the Odyssey did not recognize the target proteins, whereas at higher loadings the system incorrectly integrated the abundance of misshaped protein bands and lost the response linearity. Expectantly, we could also quantify proteins from gel lanes with no visible protein bands (Fig. 5C, 5E); for better visualization we also present the expanded gel image showing the four most diluted samples of the corresponding calibration plot (supplemental Fig. S17).
Contrary to WB that is often pestered with false positives, MS Western seems to be more prone to false negatives,  9,12,13,14,15,16,17 and GAPDH from lanes 5,6,7,8,9,12,13,14,15,16,17,18,19 from the gel image in panel B as reported by the Odyssey. E, Molar amount of TUBA1A and CP03 quantified by MS Western from slabs excised from lanes 2, 3,4,5,6,7,8,9,11,12,13,14,16,17,18  particularly if overwhelming background abolishes the ionization of proteotypic peptides and/or if target proteins are extremely low abundant. Nevertheless, we were able to quantify proteins at the subfemtomole to hundred attomole range in complex biological extracts. supplemental Fig. S18 presents calibration plots for subfemtomole quantification of BSA spiked into a total protein extract from E. coli. supplemental Fig. S19 presents the attomole quantification of 229 kDa transmembrane protein axotactin in two biological conditions from a total extract of Drosophila eye using 264 kDa chimera CP04 (supplemental Fig. S7, S8 and supplemental Table S4). The figure provides BPC and XIC traces as well as FT MS spectra of light and heavy forms of the two axotactin peptides and of five reference peptides from BSA acquired in the same quantification experiment. We also provide the ratios of relative abundances of light and heavy peptides suggesting that even at the hundred attomoles level protein quantification with both peptides was concordant.
We note that MS Western workflow does not enhance the detection sensitivity compared with conventional GeLC-MS/MS and might fail to quantify very low abundant proteins that might still be detectable by classical Western. Although in MS Western a linear calibration plot could be extended down to 100-attomoles of a gel separated protein standard (supplemental Fig. S20), it is nevertheless advisable to always start with preliminary GeLC-MS/MS experiments before designing CPs to make sure that target proteins are detectable and to select the optimal constellation of proteotypic peptides.
Absolute Quantification of Histones in Zebrafish Embryos-Histones make up the basic unit of chromatin, the nucleosome. Each nucleosome consists of 147 base pairs of DNA wrapped around a histone octamer comprising two copies of each of the four core histones: H2A, H2B, H3, and H4. Embryos inherit histones from the mother (55) and previous WB analyses suggested that, despite embryo growth, histones level is stable during the early stages of development in both Xenopus (56) and zebrafish (57), but increases rapidly upon genome activation. We reasoned that predictable stoichiometry and time course of their total content make embryonic histones a good model for validating MS Western in in vivo experiments.
We employed MS Western to determine the molar content of H2A, H2B, H3, and H4 in zebrafish embryos at ten developmental stages ranging from 1-cell stage to Shield stage. Zebrafish embryos (1-cell stage n ϭ 5; otherwise n ϭ 10) were collected and (except for 1-cell stage) manually deyolked (58). Proteins extracted from deyolked embryos were subjected to SDS-PAGE. Slabs corresponding to the M r range of ca. 5 to 25 kDa that contained all core histones were excised from each gel and histones were quantified using the bands of CP02 and BSA. We observed that ratios of relative abundances of light and heavy forms of proteotypic peptides (Fig.  6A) varied by less than 10% in all embryogenesis stages and concluded that endogenous peptides were not harboring PTMs. Accurate (CV Ͻ10%) absolute quantification of four histones (Fig 6B; supplemental Table S9) revealed that, in contrast to previous reports, histones content steadily increased already during the early stages of development (59). This suggested that in zebrafish embryos mothers deposit both histone proteins and corresponding mRNA that are translated before the onset of zygotic transcription around the 1000-cell stage. As expected, after genome activation, histones content increased more rapidly.
Molar quantities of individual histones were consistent with the expected equimolar stoichiometry (Fig. 6C). At the very early stages of development the histones stoichiometry slightly, yet consistently deviated from equimolar ratios, however that were closely approached at the later stages. We note that MS Western quantification only reflected the total content of histones, irrespectively if they were assembled into a nucleosome or associated with chaperones (55,60).
We also quantified histones by the Odyssey (Fig. 6D) and compared it to MS Western (supplemental Fig. S21). We found that the quantification by Odyssey was generally inconclusive and stoichiometry between individual histones was nowhere close to the expected equimolar ratios. This, once again, highlighted that WB quantification is extremely sensitive toward the quality of antibodies and protein standards.
Taken together, MS Western enabled precise and consistent quantification of the molar amounts of four core histones at the subpicomole per embryo level directly from total protein extracts and revealed their unexpected individual dynamics during early embryogenesis.
Conclusions and Perspectives-We argue that MS Western is a practical and technically simple solution for the accurate multiplex targeted absolute quantification of proteins. In MS Western workflow SDS-PAGE circumvents the limited solubility of both target and chimera proteins; it also removes interfering buffers and detergents, including SDS. Preseparation of total protein extracts improves the dynamic range and sensitivity of quantification. Protein quantification relies on multiple proteotypic peptides and the concordance between relative abundances of matching pairs of heavy and light peptides provides independent validation of the quantification consistency. However, one conceptual limitation of MS Western is that it is difficult to predict if a low abundant protein (e.g. a transcription factor) could be detectable by GeLC-MS/MS. In our experience the detection of proteins by classic Western blotting or projecting their abundances from transcriptomics did not substitute preliminary analyses by GeLC-MS/MS and it is advisable to establish the scope of detectable proteins before planning systematic MS Western experiments. However, once target proteins have been detected, their accurate absolute multiplexed quantification becomes a straightforward technical task.
In the future it would be interesting to explore if a single, reasonably large CP could cover all essential members of metabolic and/or signaling pathways and allow us to relate their molar abundances to molar concentrations of corresponding metabolites. It would also be intriguing to explore if CP could be used as templates to incorporate site-specific post-translational modifications by some chemical or genetic means.
Acknowledgments-We thank the members of AS, NV, and FB laboratories for useful discussion and expert technical support. We are particularly grateful for Dr. Henrik Thomas (MPI CBG) for developing useful data processing tools.