Tandem mass spectra were extracted from the Xcalibur data system format. (.RAW) and charge state assignment was performed using in house software

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1 Supplementary Methods Software Interpretation of Tandem mass spectra Tandem mass spectra were extracted from the Xcalibur data system format (.RAW) and charge state assignment was performed using in house software (RAW_Xtractor). Tandem mass spectra were interpreted by SEQUEST 1, which was parallelized on a Beowulf cluster of ~35 computers 2 and results were filtered, sorted, and displayed using the DTASelect program. 3 Searches were performed against the combined human, mouse, rat, and yeast databases from RefSeq and the wormpep115 database from Extraction of Chromatograms After filtering the results from SEQUEST using DTASelect (+1 > 1.5, +2 > 2.1, +3 > 3.1, CN > 0.08), ion chromatograms were generated from either MS or tandem mass spectra using a modified version of a program previously written in our lab (RelEx). 4 This software is available from the authors for individual use and evaluation through an Institutional Software Transfer Agreement (see for details). In all cases, the Xcalibur.RAW files were used to generate chromatograms from the m/z range surrounding both the unlabeled and labeled precursor or fragment ions. The elemental compositions and corresponding isotopic distributions for both the unlabeled and labeled peptides and all potential +1 fragment ions were calculated. This information was then used to determine the appropriate m/z range from which to extract ion

2 intensities, which included all isotopes with greater than 10% of the calculated monoisotopic peak abundance. For extraction from tandem mass spectra, the peptide precursor m/z was used to identify the proper mass window containing the appropriate tandem mass spectrum for both the unlabeled and labeled peptides. Often, the fragmentation patterns for these peptides were in different windows, but in some instances (i.e., depending on the mass shift, charge state, and size of mass window employed) both unlabeled and labeled fragmentation patterns appeared in the same tandem mass spectrum. Regardless, the ion intensities from all predicted +1 fragment ions were summed and reconstructed ion chromatograms were stored in tab delimited text files with a.chro extension. Calculation of Peptide and Protein Ratios RelEx was used to calculate peptide ion intensity ratios for each pair of extracted ion chromatograms (i.e., for each.chro file). This program has already been described in detail 4, so it will only briefly be discussed here. The heart of the program is a linear least squares correlation that is used to calculate the ratio (i.e., slope of the line) and closeness of fit (i.e., correlation coefficient (r)) between the data points of the unlabeled and labeled ion chromatograms. Measured peptide ratios were then sorted by protein locus and outliers, as determined by both a Dixon s Q-test and Chauvenet s criteria 5, were thrown out. Protein ratios were then calculated from the mean of remaining peptide ratios. Chromatogram Filtering Poor quality chromatograms were filtered using a multi-step filtering process implemented in RelEx. First, chromatograms were required to have a sufficient signal to noise to accurately quantify the mixtures analyzed by requiring the larger peaks (i.e.,

3 light for our standard mixtures) have a signal to noise of at least 3.5 for the 1:1 mixture, and 7.5:1 for the 10:1 mixture. For the C.elegans samples, we required the larger peak (i.e., light or heavy ) have a signal to noise of at least 5:1. Next, we required chromatographic pairs to have a correlation coefficient between unlabeled and labeled signals of at least Finally, we used the statistical methods (i.e., Q-test, and Chauvenet s criteria) already described above to remove outliers when calculating protein ratios from the average of all peptide ratios. Cross correlation algorithm for calculation of peptide molecular weight from tandem mass spectra We have developed an approach to identify the molecular weight of a peptide ion directly from its corresponding tandem mass spectrum using a cross correlation function. In general, this technique can be successfully applied to a variety of tandem mass spectra; but, tandem mass spectra with charge states > +2, poor signal to noise, or unusual fragmentation are less successful. We have shown that the molecular weight can be calculated within ±3 amu for approximately 70-85% of tandem mass spectra obtained from a trypsin digested yeast whole cell lysate. The algorithm has also shown potential for use as a spectral quality filter. This strategy can be used to identify the precursor ion for tandem mass spectra acquired using large ion selection windows in Data-Independent Collision Induced Dissociation (DICID). In essence, a mass spectrum is compared to a reversed copy of the same spectrum using a cross correlation algorithm. In this manner, the correlation score typically maximizes when complementary b and y ions overlap. Because complementary b and y ions are formed from the cleavage of the same amide bond, their summation is equal to

4 the molecular weight of the peptide + 1. By calculating the offset between complementary b and y ion pairs, it is therefore, possible to determine the precursor mass. This technique has been successfully applied to a variety of spectra of different charge states and varying overall quality and typically results in a precursor mass calculation within 3 of the true value. Methods Preparation of C.elegans Samples Eggs were prepared by bleaching ( % NaOCl, 0.75N KOH) gravid adult worms at the 91 th hour. Both samples were washed thoroughly with the standard M9 buffer and resuspended in the lysis buffer (20 mm HEPES ph 7.4, 10 mm KCl, 1.5 mm MgCl 2, 1 mm DTT, 1x EDTA free protease inhibitor cocktail (Roche)). Eggs or young adults were lysed on ice in a Dounce homogenizer (KONTES 20) with 100 strokes and centrifuged at 1000g for 10 min at 4 C. The 14 N Eggs and 15 N young adults were mixed at a 1:1 protein ratio and subjected to a 100,000g spin at 4 C for 1 h to yield a soluble fraction and a membrane fraction. The soluble fraction was then subjected to methanol/chloroform precipitation, and the precipitated protein was resuspended in 100mM Tris ph 8.5, 8 M urea. Protein Digestion Before digestion, protein mixtures were denatured with solid urea (final concentration of 8M in 100mM Tris ph 8.5), reduced with TCEP (Sigma, 3 mm final concentration for 20 min. at room temp.), and alkylated with iodoacetamide (Sigma, 10 mm final concentration for 30 min at room temp.). Digestion was then performed using

5 a previously described protocol. 6 The resulting peptide mixture was acidified with formic acid (3%). MudPIT This approach has been described in detail by several authors 6-9, so it will only briefly be detailed here. A three phase microcapillary column was constructed and equilibrated with 5% acetonitrile / 0.1% formic acid for ~30 min before the peptide mixture was loaded. For analysis, the microcolumn was positioned in-line with a Surveyor autosampler and Surveyor quaternary HPLC pump (ThermoElectron) directly in front of the heated capillary opening of either an LCQ-Deca or LTQ ion trap mass spectrometer (ThermoElectron). Peptide mixtures were loaded on-line using the autosampler and analyzed using a six or twelve step separation procedure. 6, 8, 10 As peptides were eluted and ionized into the mass spectrometer, acquisition of tandem mass spectra was repeated continuously during the course of the analysis. All tandem mass spectra were collected using a normalized collision energy of 35% and only 1 µscan was acquired (i.e., no signal averaging was employed). Typical instrumental parameters included a maximum injection time of 100 ms, an MS/MS AGC target of 10,000 ions, and a spray voltage of 2.4kV. Scan Sequences and Search Parameters Qualitative Analysis: When data-dependent acquisition was employed, a cycle consisting of one full scan mass spectrum ( m/z) followed by three tandem mass spectra was repeated throughout the analysis, and an isolation window of 3 m/z was used. The dataindependent scheme employed consisted of 100 MS/MS scans interrogating the mass

6 range between m/z (i.e., each window had an isolation width of 10 m/z) and the scan cycle time was ~35s. Each sample was separated by a 4hr reverse phase gradient and analyzed on an LTQ mass spectrometer. Acquired tandem mass spectra were then searched against the combined human, mouse, rat, and yeast databases from Refseq using SEQUEST (peptide mass tolerance = 10, enzyme specificity = trypsin), and filtered using default DTASelect criteria (i.e., +1 > 1.5, +2 > 2.1, +3 > 3.1, CN > 0.08, min. # peptides = 2, tryptic status = half). Quantitative Analysis of Yeast Lysates: The data-independent acquisition scheme consisted of 1 MS scan followed by 20 MS/MS scans interrogating the mass range between m/z (an isolation width of 10 m/z). Each scan cycle was completed in ~4s. Acquired tandem mass spectra were then searched against the yeast-orfs database from Refseq. The MS scan was acquired entirely for chromatogram comparison purposes and no data-dependent acquisition was employed. Quantitative Analysis of C.elegans: The data-independent acquisition scheme consisted of 34 MS/MS scans interrogating the mass range between m/z (an isolation width of 15 m/z). Each scan cycle was completed in ~10s. Acquired tandem mass spectra were then searched against the wormpep115 database from using SEQUEST (mass accuracy = 15 amu, enzyme specificity = trypsin). Search results were then filtered with default DTASelect parameters (i.e., +1 > 1.8, +2 > 2.5, +3 > 3.5, CN > 0.08, and at least two peptides per locus).

7 Western Blots The PNS fractions of 14 N Eggs and 15 N young adults were prepared as above, centrifuged at 100,000g for 1 h at 4 C, and the supernatants were collected for Western blotting. A total of 10 µg proteins from 14 N Eggs or 15 N young adults were separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted for SQV-4, CRT-1, and CYP-5. The immunoreactive bands were visualized with SuperSignal, an enhanced chemiluminescent substrate (Pierce).

8 References 1. Eng, J.K., McCormack, A.L. & Yates, J.R., III An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry 5, (1994). 2. Sadygov, R.G. et al. Code developments to improve the efficiency of automated MS/MS spectra interpretation. Journal of Proteome Research 1, (2002). 3. Tabb, D.L., McDonald, W.H. & Yates, J.R., III DTASelect and Contrast: Tools for Assembling and Comparing Protein Identifications from Shotgun Proteomics. Journal of Proteome Research 1, (2002). 4. MacCoss, M.J., Wu, C.C. & III, J.R.Y. A correlation algorithm for the automated analysis of quantitative shotgun proteomics data. Analytical Chemistry submitted (2003). 5. Taylor, J.R. An introduction to error analysis: the study of uncertainties in physical measurements. (University Science Books, Mill Valley, CA; 1982). 6. McDonald, W.H., Ohi, R., Miyamoto, D.T., Mitchison, T.J. & Yates, J.R. Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. International Journal of Mass Spectrometry 219, (2002). 7. Link, A.J. et al. Direct analysis of protein complexes using mass spectrometry. Nature Biotechnology 17, (1999).

9 8. MacCoss, M.J. et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proceedings of the National Academy of Sciences of the United States of America 99, (2002). 9. MacCoss, M.J., Wu, C.C. & Yates, J.R., III Probability-Based Validation of Protein Identifications Using a Modified SEQUEST Algorithm. Analytical Chemistry 74, (2002). 10. Washburn, M.P., Wolters, D. & Yates, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnology 19, (2001).