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 sub-femtomole level in whole cell or tissue lysates without metabolic or chemical labelling and without using specific antibodies. MS Western relies upon GeLC-MS/MS and quantifies proteins by in-gel co-digestion with an isotopically labelled 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.


INTRODUCTION
Despite well-known technical limitations and numerous application pitfalls, Western blotting (WB) 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 towards 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 high-end 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 labelling 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 unlabelled cell and tissue lysates that combines sample preparation versatility of conventional WB with the specificity, accuracy and sensitivity of LC-MS/MS.

Chemicals and reagents
All reagents were of the analytical grade or better quality. LC-MS grade solvents were purchased from Fisher Scientific (Waltham, MA); formic acid (FA) from Merck (Darmstadt, Germany), Complete Ultra Protease Inhibitors from Roche (Mannheim, Germany); Trypsin Gold, mass spectrometry grade, from Promega (Madison, USA); restriction enzymes and buffers from New England BioLabs (Ipswich, MA); benzonase from Novagen (Gibbstown, NJ); other common chemicals and buffers were from Sigma-Aldrich (Munich, Germany). Pre-cast 4 to 20 % gradient 1mm thick polyacrylamide mini-gels were from Anamed Elektrophorese (Rodau, Germany).

Protein standards and amino acids
Protein standards: bovine serum albumin (BSA), glycogen phosphorylase (GP), alcohol dehydrogenase (ADH), enolase (ENO) and ubiquitin (UBI) were purchased as a lyophilized powder from Sigma-Aldrich. Their purity was checked by 1D SDS PAGE and by amino acid analysis (Functional Genomics Centre Zurich, Switzerland). Ampoules of Pierce BSA standard and of recombinant human histones were purchased from Thermo Fisher Scientific (Waltham, MA) and from New England BioLabs (Ipswich, MA), respectively. Amino acids were from AppliChem (Darmstadt, Germany); isotopically labelled 13 C 6 15 N 4 -L-arginine and 13 C 6 -L-lysine were from Silantes (Munich, Germany).

Monitoring the kinetics of in-gel digestion of HeLa proteins
Protein extracts of ca. 1× 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. Upon SDS PAGE, gel slabs whose Mr 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 analysed 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 upon 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 minutes and subjected to 1D SDS PAGE. A single gel slab containing 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 = 5 for H3 and H2B; n=10 for H2A and H4). Embryos were processed as described above and total protein extracts were subjected to 1D SDS PAGE. Proteins were blotted onto a nitrocellulose membrane (GE Life Sciences). Primary antibodies (Supplementary Table S8) were incubated at room temperature for 1 hour or at 4°C overnight; secondary antibodies (Supplementary 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 homologues.

Data deposition
Proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE (33) partner repository with the dataset identifier PXD005654 and doi: 10.6019/PXD005654. Temporary login for reviewers is provided under username: reviewer55654@ebi.ac.uk and password: HgrBxHbm.

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 labelled 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 lanes separating crude protein extracts.
In the same way, we further selected proteotypic peptides from two reference proteinsin 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 degradation 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 ( Supplementary 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 ( Supplementary Fig. S2, S4, S6 and S8) and incorporated more than 99.5% of heavy arginine and lysine residues ( Supplementary 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 QconCAT 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 interlaboratory inconsistency. Instead, MS Western utilizes a simple workaround solution that requires no CP purification (Fig. 2).
To quantify proteins of interest a cell or tissue lysate was subjected to 1D SDS PAGE. Gel  Table S1). GeLC-MS/MS of its gel band yielded 100% sequence coverage (Supplementary Fig. S2B) and confirmed ≥99.5% isotopic labelling efficiency (Supplementary Fig. S9).
Gel bands of each of these five target proteins were co-digested with the band of CP01. The extracted peptides were analyzed by LC-MS/MS and relative abundances of light (normalized to all light) and heavy (normalized to all heavy) proteotypic peptides were compared ( Supplementary Fig.   S10). We observed that within 31 peptide pairs the relative abundances varied by less than 5% ( Supplementary Fig. S10), despite light and heavy peptides originated from structurally different chimera and endogenous proteins. Indeed, prior to 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 pre-separation of crude extracts by SDS PAGE, only a small fraction of a background proteome was co-digested together with target protein(s). In line with previous findings (32), we observed no 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 (Supplementary 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 ( Supplementary   Fig. S3, S4 and Supplementary 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 (Supplementary 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) peptides mismatched because of trypsin miscleavage caused by the presence of flanking dibasic amino acid residues in the sequences of endogenous proteins (45,(50)(51)(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 co-digested each at its own kinetics and if digestion rate matching was required for accurate absolute quantification?
To this end, we monitored in-gel digestion of α-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 Mr, co-digested with bands of 72 kDa chimera protein CP03 ( Supplementary Fig. S5, S6 and Supplementary 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, B and C. In the HeLa extract the abundance of TUBA and CAT differed by ca 100-fold (Fig. 4B, C) and they were digested together with ca 1100 comigrated background proteins. As a consistency check, we 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 hr, while MS Western protocol relies on overnight digestion. Interestingly, not all proteotypic peptides were produced at the same rate (Fig. 4D, E). The ratio of relative abundances of light and heavy forms of most peptides plateaued at the expected value of 1.0 already after 3 hr, consistently with the kinetic plots in Fig. 4B, C. However, two peptides showed deviating trends. The ratio of relative abundances of peptide QTALVELLK was lower than expected (Fig. 4D, Supplementary Figure S13) because of miscleavage of native BSA (see also Fig. 3A). Consistently with earlier reports on the specificity of trypsinolisys of peptides flanked with successive Arg, Lys residues (45,50,51,54) the yield of QTALVELLK did not improve at extended digestion times, while 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 hours (Fig. 4E, Supplementary Figure 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  Supplementary 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 (Supplementary 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. 5 B, C). 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 co-digested with each sample and therefore its constant abundance evidenced the analyses consistency. Compared to the Odyssey, MS Western showed at least 60-fold better sensitivity (down to 1.5 fmol of TUBA1A) along with higher dynamic range (>16000-fold) and excellent linearity (r 2 = 0.9983) (Fig.5E). In the middle of the plot (Fig.5D) the Odyssey also showed good linearity towards both GAPDH and TUB1A/4A. However, at lower loadings the Odyssey did not recognize the target proteins, while at higher loadings the system incorrectly integrated the abundance of irregular-shaped protein bands and lost the response linearity. Expectantly, we could also quantify proteins from gel lanes with no visible protein bands (Fig. 5C, E); for better visualization we also present the expanded gel image showing the four most diluted samples corresponding calibration plots (Supplementary Fig. S17).
Contrary to WB that is often pestered with false positives, MS Western seems to be more prone to false negatives, 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 sub-femtomole to hundred attomole range in complex biological extracts. Supplementary Fig.S18 presents calibration plots for sub-femtomole quantification of BSA spiked into a total protein extract from E.coli. Supplementary 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 ( Supplementary Fig. S7, S8 and Supplementary 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 were concordant.
We note that MS Western workflow does not enhance the detection sensitivity compared to 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=5; otherwise n=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 Mr 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 harbouring PTMs. Accurate (CV <10%) absolute quantification of four histones (Fig 6B; Supplementary 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 ( Supplementary 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 towards 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 sub-picomole 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. Pre-separation of total protein extracts improves the dynamic range and sensitivity of quantification. Protein quantification relies on multiple proteotypic peptides and the concordance between the relative abundances of matching pairs of heavy and light peptides provides independent validation of the quantification consistency. However, one conceptual limitation of MS  . Sequences of proteotypic peptides (schematically shown as boxes) from each target protein (colour-coded) are insilico 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. The CP is quantified by comparing the abundances of XIC peaks of unlabelled (from BSA reference) (E) and labelled (from the CP) (F) peptides (G). Next, the amount of target protein is inferred from the ratio of abundances of XIC peaks of matching unlabelled (from the target protein) (H) and labelled (from the CP) (I) proteotypic peptides (J) and from the amount of CP. For clarity, only two (out of several) matching pairs of labelled / unlabelled peptides are shown. A solubilised cell pellet in 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 as an example.    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 from the gel in panel C. Lanes 1,10,11,20 and lanes 1, 10, 15 and 20 are MW markers in panels B and C, respectively.