Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis.


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In terms of both high sensitivity and accuracy, mass spectrometry (MS) is becoming one  To validate the newly designed tags, we tried to measure protein mixtures with known 1 4 1 concentration using the MS-QBiC method based on 34 quantification tags. As a protein 1 4 2 mixture, we used a commercially available equimolar tryptic peptide mixture from six 1 4 3 proteins including bovine cytochrome C (12 kDa), chicken lysozyme C (16 kDa), yeast 1 4 4 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 16, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 6 alcohol dehydrogenase 1 (37 kDa), bovine serum albumin (69 kDa), bovine Digest, equimolar, Thermo Scientific). A total of 34 target peptides was selected for 6 1 4 7 proteins quantification (Data S1 and Table S3). For the selection of the target peptides, 1 4 8 we focused on those composed of 6 to 20 amino acids without Met and Cys. Also, the 1 4 9 peptide sequences that may cause miss-cleavages, such as KP, RP, KK, and RR were 1 5 0 avoided. We note that the original quantification tag, LVTDLTK, was not used for this reactions, and then, all 34 templates were added to the PURE system and peptides were including tryptic digestion. The yield of each MS-QBiC peptide was measured by LC-MS analysis using Orbitrap mass spectrometer in full-scan mode (Fig. 2a). The measurement was based on 1 6 2 the light/heavy ratio of the quantification tags and it showed that all 34 MS-QBiC 1 6 3 peptides were successfully synthesized. The yields from 5 µl PURE reaction mixture 1 6 4 ranged between 11 and 126 fmol and the total amounts were 2 pmol. They were within 1 6 5 the range for the MS-based quantification, although some peptides, such as 1 6 6 YVVDTSK*, AWSVAR*, LVNELTEFAK*, YYGYTGAFR*, and LWSAEIPNLYR*, showed low yield (asterisks represent labeled lysine or arginine). We note that 1 6 8 additional synthesis of peptides can compensate for the low yield of specific peptides. When YVVDTSK*, LVNELTEFAK*, and LWSAEIPNLYR* were synthesized 1 7 0 separately, the yield from 1 µl of the PURE reaction were 200, 270, and 120 fmol,  Subsequently, target peptides in a six proteins mixture were quantified based on the light/heavy ratio of the target peptides by LC-MS analysis (Fig. 2b, red). We also 1 7 5 performed selected reaction monitoring (SRM) using triple quadrupole mass 1 7 6 spectrometer and found that the quantification results were almost similar with those with LC-MS analysis (Fig. 2b, orange). Further validation was performed by shuffling the quantification tags (Table S4) and target peptides were quantified with both LC-MS CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made and SRM. We found that the quantification results did not vary according to the used 1 8 1 quantification tags (Fig. 2b, blue and green). product of YIPGTK (Fig. S1a). Also deamidated products originated from NYQEAK 1 8 6 and QQDDFGK were found (Fig. S1b, c). NYQEAK also showed early retention time, suggesting it is very hydrophilic and not sufficiently retained by the column (Fig. S1b).
As a result, these peptides were quantified at relatively low values (Fig. 2b). After excluding the values of these three peptides, average of quantification values of 1 9 0 cytochrome C, lysozyme C, alcohol dehydrogenase 1, serum albumin, serotransferrin, 1 9 1 and beta-galactosidase were 62, 102, 103, 113, 95, and 86 fmol, respectively, which 1 9 2 were within the same order of magnitude as the amounts of added proteins (167 fmol). proteins. Quantifying ribosome composition is crucial for understanding its biogenesis 1 9 8 [17,18]. Also it might be important because the ribosome stoichiometry is suggested to 1 9 9 be variable depending on tissue types, physiological conditions, and aging, which might  A total of 68 peptides were designed (Data S2 and Table S5) and two sets of the 2 0 2 PURE reaction were performed where 34 peptides were synthesized in each reaction.  the peptides were within the same order of magnitude as the amount of added 30S 2 1 0 subunit (Fig. 3a). Five peptides marked with asterisks (ISELSEGQIDTLR from uS13, 2 1 1 AIISDVNASDEDR from uS14, WNAVLK from uS14, GPFIDLHLLK from S19, and 2 1 2 ENEPFDVALR from S21) were less than 30% compared to the added 30S subunit these peptides (Fig. S2), suggesting these peptides are not suitable for the quantification. After excluding the values of these five peptides, protein amounts were calculated as the 2 1 6 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (Fig. 3b). The data showed that 2 1 7 all proteins were within the same orders of magnitude, suggesting equimolar amount of 2 1 8 proteins are included in the prepared 30S subunit. is indispensable to select appropriate target peptides, which are effectively ionized and 2 2 5 detectable on MS ("proteotypic") and quantitatively reliable ("quantotypic") [22,23]. computationally and experimentally that takes much time and effort [24,25], and then, that preparing all target peptides as a SIL form is difficult for financial reasons. Furthermore, there is a problem that the practical screening is not always applicable 2 3 4 when acquiring standard proteins are difficult.  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made and therefore enables short and high-throughput analysis [27]. After processing the peak 2 5 3 data and calculation, some peptides show outliers due to miss-cleavages, noisy peaks, or 2 5 4 missing peaks. Those discordances can be corroborated by analyzing back the full scan 2 5 5 data and the outliers will be eliminated accordingly. Whole processes will be completed  cell-free synthetic heavy peptides and quantification of target peptides in samples.

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Furthermore, we showed that it is possible to increase the multiplexity by using multiple systems, e.g., signal transduction networks. Preparation of plasmid encoding an original quantification tag (LVTDLTK) was already original template plasmid at 37 °C overnight. Using the remaining PCR products, E.  Non-labeled quantification tags were synthesized by Fluorenylmethyloxycarbonyl 2 9 7 (Fmoc) chemistry using a peptide synthesizer SyroWave (Biotage, Uppsala, Sweden). Chemical Industries. Piperidine was purchased from Nacalai Tesque (Kyoto, Japan).

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Fmoc deprotection was performed using 40% piperidine/DMF for 3 + 12 min. Coupling  The peptides were cleaved from the resin with reagent K (82.5% TFA, 5% phenol, 5% peptides were precipitated and washed with pre-cooled diethyl ether, and dried. The   Using the plasmids encoding each quantification tag as templates, DNA templates for 3 2 2 MS-QBiC peptide synthesis were amplified by PCR using Taq DNA polymerase (NEB) 3 2 3 with a T7 promoter primer (5′-GGGCCTAATACGACTCACTATAG-3′) as a forward 3 2 4 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 16, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 1 1 primer and appropriate reverse primers listed in Table S4 and Table S6. PCR product  Enzymatic digestion was performed basically according to a phase transfer surfactant Two types of mass spectrometers were used. One was an Orbitrap mass spectrometer Velos Pro, Thermo Scientific), which was used for a high-resolution full-scan analysis.

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The other was a triple quadrupole mass spectrometer (positive mode, Q1 and Q3 Vantage, Thermo Scientific), which was used for SRM analyses. Both of them were 3 5 6 equipped with a nanospray ion source (Nanospray Flex, Thermo Scientific) and a 3 5 7 nano-LC system (UltiMate 3000, Thermo Scientific). Peptides were concentrated using and then separated using a nano capillary column (0.1 × 150 mm, 3 µm, C18, Nikkyo 3 6 0 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Technos) at a flow rate of 500 nL/min using two mobile phases A (0.1% formic acid) 3 6 1 and B (acetonitrile and 0.1% formic acid). For the analysis of tryptic six protein mix, we 3 6 2 used a gradient of 5% B for 5 min, 5-35% B in 65 min, 35-90% B in 1 min, and 90% B . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 16, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021