Online Appendix 3. Supplemental Information Methods...of at least one pair-wise comparison (obese vs. lean, diabetic vs. lean, and obese vs. diabetic). Finally, the proteins that were

Differential Human Skeletal Muscle Proteome in Insulin Resistance

©2009 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/cgi/content/full/db09-00214/DC1

Online Appendix 3. Supplemental Information Methods The complete methods used in these studies are described below.

Subjects. Lean nondiabetic control subjects, obese nondiabetics, and patients with type 2 diabetes (n=8 each) were studied. Studies were approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio and Arizona State University. After giving informed, written consent, each volunteer underwent a medical history, physical examination, screening laboratory tests and a 75 g oral glucose tolerance test. The lean control and obese nondiabetic subjects had no family history of diabetes and had normal glucose tolerance. The participants also were instructed to not depart from their regular diet three days before study, and not to engage in exercise for two days before the study. Participants reported less than two sessions of exercise per week and were considered to be sedentary. Patients with type 2 diabetes were excluded if they were treated with metformin or a thiazolidinedione. If treated with a sulfonylurea, treatment was withheld for three days before the study. Patients treated with insulin had the last insulin dose on the previous day withheld. Following a 10 to 12-h overnight fast, subjects came to the General Clinical Research Center (GCRC) at the University of Texas Health Science Center at San Antonio or Clinical Research Unit (CRU) at Arizona State University for screening tests. Subjects returned to the GCRC or CRU at 8 A.M. after an overnight fast for a 120-min euglycemic-hyperinsulinemic (80 mU·m-2·min-1) insulin clamp study, as described (1; 2). Percutaneous biopsies of the vastus lateralis muscle were taken under local anesthesia under basal conditions, immediately frozen in liquid nitrogen, and stored frozen until they were homogenized as described (3).

Gel electrophoresis and mass spectrometry analysis. Muscle lysates proteins (60 µg total protein) were separated on 4-20% linear gradient SDS polyacrylamide gels, and processed for mass spectrometry as described (4; 5) and as outlined in Supplemental Figure 1. Each lane was cut into 20 separate slices. Gel pieces were treated with trypsin to digest proteins, the resulting mixture desalted, and subjected to HPLC-MS/MS. Therefore, since 24 subjects were analyzed, a total of 480 HPLC-MS/MS runs were performed. HPLC-ESI-MS/MS was performed on a hybrid linear ion trap (LTQ)-Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer (LTQ FT, Thermo-Fisher, San Jose, CA) (4; 5). Protein assignment (99% confidence) and gene ontology annotation were performed as described (4; 5). Tandem mass spectra were extracted from Xcalibur “RAW” files and charge states were assigned using the Extract_MSN script (Xcalibur 2.0 SR2, Thermo-Fisher, San Jose, CA). Charge states and monoisotopic peak assignments were verified using DTA-SuperCharge, part of the MSQuant suite of software (msquant.sourceforge.net), before all “DTA” files (corresponding to each gel slice from a single gel lane) were combined into a single Mascot Generic Format (GF) file that represented the data from a single subject. The fragment mass spectra were then searched against the IPI-HUMAN_v3.28 database (68,020 entries, hhtp://www.ebi.ac.uk/IPI) using Mascot (Matrix Science, London, UK; version 2.2). The false positive discovery rate was determined by selecting the option to search the decoy randomized database, and using the two-peptide criterion for protein assignment was found to be less than 1%. The search parameters that were used were: 10 ppm mass tolerance for precursor ion masses and 0.5 Da for product ion masses; digestion with trypsin; a maximum of two missed tryptic cleavages; variable modifications of oxidation of methionine and phosphorylation of serine, threonine and tyrosine. Probability assessments of peptide and protein assignments were made through use of Scaffold (version Scaffold-01_06_19,



Proteome Software Inc., Portland, OR). Only peptides with > 95% probability, based on Scaffold analysis, were considered. Criteria for protein identification included detection of at least 2 unique peptides and a probability score of > 99%. Multiple isoforms of a protein were reported only if they were differentiated by at least one unique peptide. Proteins that contained identical peptides and could not be differentiated based on MS/MS analysis alone were grouped. The spectra assigned to shared peptides were excluded from quantification using NSAF.

Gene Ontology annotation of human proteins was downloaded from Gene Ontology Annotation (GOA) Databases (http://www.ebi.ac.uk/GOA, version 55.0). This GOA human database contains 33,731 distinct proteins and 172661 GO associations. In addition, GO hierarchy information (version: 52) was downloaded from www.geneontology.com. Human GO associations and GO hierarchy information were assembled into a new database by an in-house program written using MATLAB (The Mathworks, Natick, MA). IPI IDs, gene names, UniProt and SwissProt IDs of identified proteins were input into the database to obtain GO associations and GO hierarchy information.

Normalized Spectral Abundance Factors. To quantify protein abundance, normalized spectral abundance factors (NSAF) were used (6-8). MS/MS spectra assigned to a protein were normalized to the length of the protein (number of amino acids), resulting in a Spectral Abundance Factor, or SAF, NumberAAuntSpectrumCoSAF = . Each SAF was normalized against the sum of all SAFs in one sample, resulting in the NSAF value. For a protein, i, the

normalized spectral abundance factor, NSAF, is calculated by ∑=

=N

iiii SAFSAFNSAF

1

, where N

is the total number of proteins detected in a sample. Thus, NSAF values allow for direct comparison of a protein’s abundance between individual runs in a fashion similar to microarray data analysis.

Reproducibility and linearity of NSAF method. To determine linearity, varying amounts of BSA tryptic digests (62 to 250 fmoles) were added to 125 fmoles of cytochrome c digest. Mixtures were analyzed by MS/MS. To assess linearity in a “real life”, complex mixture of proteins, muscle biopsy lysates were used. If NSAF provides an accurate reflection of the proportion any particular protein in a complex protein sample, such as a muscle lysate, then the NSAF for any given protein should be equivalent, no matter how much total protein is analyzed. To test this 30, 60 and 120 µg protein from the same muscle lysate were loaded onto a gel in three separate lanes. Proteins were assigned and NSAF values were calculated as described above. NSAF values for each of the proteins that were assigned in common to each of three lanes lane were plotted against each other. If the values were equal, it would be expected that a linear regression performed on these points would have a slope of 1.0 and a Y intercept of 0. To test reproducibility, a muscle biopsy from one subject was divided into three sections. The three biopsy specimens, which theoretically should contain the same proteins in the same proportions, were homogenized, 60 µg of lysate proteins were run in three separate lanes on a 4-20% linear gradient SDS gel, the lanes were cut into slices, the slices were digested with trypsin, digests were analyzed by HPLC-MS/MS, and spectra thus obtained were used to assign proteins and calculate NSAF values.

Single protein analysis. Although a large number of proteins were assigned in at least one of 24 subjects who were studied, a series of filters were used to narrow the number of proteins that were used in comparisons among groups. This approach is diagrammed in Figure 1. First, NSAF values for each assigned protein were determined for each member of each group of subjects. In individuals for whom no spectra for a protein were observed, the NSAF value for



that protein for that subject was set to zero, and zeroes were included in all analysis of single protein abundance differences. The first filter applied was to exclude all proteins from further analysis that were not detected in at least half (12 of 24) of the subjects. For example, a protein found in 8 lean, 3 obese, and 1 diabetic subjects (12 of 24) would be included in the analysis. The next step was to calculate average NSAF values for each group for each protein that was present in at least half the subjects. The second filter applied was for proteins that differed in abundance by a factor of two (increased by two-fold or decreased by half) between the averages of at least one pair-wise comparison (obese vs. lean, diabetic vs. lean, and obese vs. diabetic). Finally, the proteins that were assigned in at least 12 of 24 subjects and were increased in abundance by a factor of two or decreased by one half were subjected to one-way analysis of variance with post hoc t-tests using StatView (SAS Institute, 1998). Because about half of these proteins did not meet the assumption of analysis of variance of homogeneity of variances, a Kruskal-Wallis nonparametric equivalent of analysis of variance also was performed using StatView. The correlation coefficient between P values for the two tests was 0.76 (P < 0.01).

Differential patterns of protein abundance among groups (protein sets). Differences in NSAF values for a set of proteins among groups were assessed as follows (Figure 2). First, for each protein in a set, an average NSAF value was calculated using all 24 values (including zeroes) from the three groups of eight. Second, each individual NSAF value for a protein was normalized, or scaled, by dividing the NSAF by the overall average NSAF value for that protein. Third, a composite variable was constructed for each subject, consisting of the average of normalized NSAF values over all proteins in the set. Finally, one-way analysis of variance was used to compare the differences in composite NSAF values among groups. Only sets that consisted of 5 or more proteins were used.

In order to better visualize patterns of differences among groups, protein abundance changes were visualized using Gene Expression Dynamics Inspector or GEDI, website http://www.childrenshospital.org/research/ingber/GEDI/gedihome.htm (9). GEDI employs an algorithm that groups individual proteins by patterns of group differences compared to an overall median. Proteins for a particular grouping that are generally lower in abundance than the global mean are depicted in shades of blue, while proteins that are increased are depicted in shades of orange and red, similar to a heat map.

Immunoblot analysis. To determine the relationship between NSAF quantification and relative protein abundance determined by immunoblot analysis, two proteins (desmin and alpha actinin-2) that differed significantly among the groups by NSAF analysis were subjected to immunoblot analysis. Only 7 of the 8 subjects in each group had sufficient muscle protein remaining to perform these analyses. Muscle protein lysates were separated by SDS-PAGE, transferred to membranes and analyzed using antibodies against alpha actinin-2 or desmin (both from Santa Cruz Biotechnology, Santa Cruz, CA). Blots were quantified by densitometry (VersaDoc 5000: Bio-Rad Laboratories, Hercules, CA). Supplemental References 1. Cusi K, Maezono K, Osman A, Pendergrass M, Patti ME, Pratipanawatr T, DeFronzo RA, Kahn CR, Mandarino LJ: Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle. J Clin Invest 105:311-320, 2000 2. DeFronzo RA, Tobin JD, Andres R: Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 237:E214-223, 1979



3. Koval JA, Maezono K, Patti ME, Pendergrass M, DeFronzo RA, Mandarino LJ: Effects of exercise and insulin on insulin signaling proteins in human skeletal muscle. Med Sci Sports Exerc 31:998-1004, 1999 4. Hojlund K, Yi Z, Hwang H, Bowen B, Lefort N, Flynn CR, Langlais P, Weintraub ST, Mandarino LJ: Characterization of the human skeletal muscle proteome by one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. Mol Cell Proteomics 7:257-267, 2008 5. Yi Z, Bowen BP, Hwang H, Jenkinson CP, Coletta DK, Lefort N, Bajaj M, Kashyap S, Berria R, De Filippis EA, Mandarino LJ: Global relationship between the proteome and transcriptome of human skeletal muscle. J Proteome Res 7:3230-3241, 2008 6. Pavelka N, Fournier ML, Swanson SK, Pelizzola M, Ricciardi-Castagnoli P, Florens L, Washburn MP: Statistical similarities between transcriptomics and quantitative shotgun proteomics data. Mol Cell Proteomics 7:631-644, 2008 7. Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP: Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res 5:2339-2347, 2006 8. Zybailov BL, Florens L, Washburn MP: Quantitative shotgun proteomics using a protease with broad specificity and normalized spectral abundance factors. Mol Biosyst 3:354-360, 2007 9. Eichler GS, Huang S, Ingber DE: Gene Expression Dynamics Inspector (GEDI): for integrative analysis of expression profiles. Bioinformatics 19:2321-2322, 2003 Supplemental Figure 1



Supplementary Tables Supplemental Table 1 is available in Online Appendix 2 Supplemental Table 2 is available in Online Appendix 3 Supplemental Tables 3‐5 are included below:



3 Supplemental Table 3. Proteins differing by a factor of at least 2 between at least two groups but not statistically significant by ANOVA.

Protein Name NSAF ANOV

A Kruskal-Wallis

# Subjects with Protein Present Total Spectra per Group

Lean Obese Diabetic P P Lean Obese Diabetic Lean Obese Diabetic PROTEIN-L-ISOASPARTATE (D-ASPARTATE) O-METHYLTRANSFERASE 0.37±0.25 1.67±0.40 1.04±0.58 0.13* 0.06 2 7 3 3 10 6 DELTA-1-PYRROLINE-5-CARBOXYLATE DEHYDROGENASE, MITOCHONDRIALPRECURSOR 0.38±0.19 1.39±0.76 0.56±0.38 0.34* 0.50 4 5 3 7 20 8 UV EXCISION REPAIR PROTEIN RAD23 HOMOLOG B 0.24±0.16 0.84±0.45 1.32±0.40 0.13* 0.09 2 4 6 3 9 14 VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 35 0.30±0.16 1.00±0.28 0.60±0.35 0.22 0.15 3 6 3 7 20 13 BIFUNCTIONAL PURINE BIOSYNTHESIS PROTEIN PURH 0.75±0.24 2.44±0.82 1.19±0.53 0.13* 0.25 6 7 5 13 40 20 RIBOSOMAL PROTEIN S6 KINASE ALPHA-3 0.19±0.16 0.57±0.20 1.10±0.39 0.08* 0.15 2 5 5 4 11 21 T-COMPLEX PROTEIN 1 SUBUNIT ALPHA 0.89±0.26 2.56±0.91 1.61±0.62 0.22* 0.15 6 6 7 14 37 22 1,4-ALPHA-GLUCAN BRANCHING ENZYME 0.55±0.28 1.59±0.59 0.91±0.36 0.25 0.43 4 5 5 12 29 17 ISOFORM I OF CALPAIN-3 0.40±0.19 1.11±0.44 0.64±0.23 0.27 0.53 4 5 5 9 23 13 PROTEASOME SUBUNIT ALPHA TYPE 2 1.35±0.61 3.76±0.89 3.48±0.76 0.07 0.06 4 8 7 9 23 21 26S PROTEASOME NON-ATPASE REGULATORY SUBUNIT 3 0.60±0.29 1.60±0.90 1.37±0.35 0.46* 0.39 4 3 6 9 23 19 26S PROTEASE REGULATORY SUBUNIT 4 0.50±0.26 1.35±0.45 0.74±0.38 0.27 0.32 3 6 4 6 14 9 AMINE OXIDASE [FLAVIN-CONTAINING] B;MONOAMINE OXIDASE B 0.46±0.20 1.22±0.26 0.80±0.32 0.15 0.16 4 7 5 8 17 11 PROTEIN CGI-38 1.01±0.50 2.57±0.88 1.68±0.76 0.34 0.43 3 5 4 6 12 18



5'-AMP-ACTIVATED PROTEIN KINASE CATALYTIC SUBUNIT ALPHA-2 0.64±0.36 1.61±0.65 0.72±0.32 0.29 0.36 3 5 4 9 24 11 MYOSIN BINDING PROTEIN C, SLOW TYPE ISOFORM 1 0.15±0.12 0.38±0.16 0.39±0.13 0.4 0.29 2 5 5 6 12 12 T-COMPLEX PROTEIN 1 SUBUNIT ETA 0.23±0.18 0.56±0.28 0.48±0.11 0.49* 0.24 2 4 6 3 8 7 ISOFORM LONG OF 14-3-3 PROTEIN BETA/ALPHA;ISOFORM SHORT OF 14-3-3 PROTEIN BETA/ALPHA 1.20±0.69 2.94±0.76 2.43±0.95 0.31 0.42 4 6 4 9 19 16 CARNITINE O-PALMITOYLTRANSFERASE 2, MITOCHONDRIAL PRECURSOR 0.40±0.21 0.97±0.35 1.27±0.44 0.22 0.23 4 6 6 6 17 23 ACTIN, CYTOPLASMIC 1;ACTIN, CYTOPLASMIC 2 0.54±0.27 1.31±0.43 1.34±0.35 0.22 0.21 4 5 7 6 13 13 ISOFORM 1 OF LEUKOTRIENE A-4 HYDROLASE 0.42±0.22 1.00±0.24 0.41±0.09 0.07* 0.08 3 7 6 6 16 7 PHENYLALANYL-TRNA SYNTHETASE BETA CHAIN 0.67±0.36 1.58±0.87 1.09±0.51 0.58* 0.66 3 5 4 10 25 16 HEMOPEXIN PRECURSOR 1.11±0.40 2.63±0.83 1.51±0.49 0.21 0.44 5 7 5 15 31 17 NADP-DEPENDENT MALIC ENZYME 0.56±0.34 1.27±0.31 1.27±0.35 0.24 0.14 3 7 7 8 19 19 187 KDA PROTEIN;COMPLEMENT C3 PRECURSOR (FRAGMENT) 1.95±0.52 4.38±1.36 2.10±0.53 0.12* 0.18 7 8 8 94 176 91 ALDEHYDE DEHYDROGENASE, MITOCHONDRIAL PRECURSOR;MITOCHONDRIAL ALDEHYDE DEHYDROGENASE 2 VARIANT (FRAGMENT) 2.17±0.39 4.81±1.93 3.15±0.71 0.31* 0.60 7 6 8 35 63 42 PRENYLCYSTEINE OXIDASE PRECURSOR 1.31±0.26 2.90±0.85 3.09±0.52 0.09* 0.05 8 6 8 19 37 41 PROTEASOME SUBUNIT BETA TYPE 4 PRECURSOR 1.13±0.44 2.48±0.69 1.97±0.52 0.25 0.16 4 6 6 9 16 14



CDNA FLJ25678 FIS, CLONE TST04067, HIGHLY SIMILAR TO PURINE NUCLEOSIDEPHOSPHORYLASE 1.64±0.52 3.55±1.73 1.76±0.90 0.44* 0.81 5 4 3 15 25 12 PROTEIN S100-A6 13.9±7.16 30.0±10.5 38.3±12.1 0.25 0.39 3 5 6 39 70 93 ALPHA-ACTININ-3 1.00±0.51 2.15±0.78 1.90±0.83 0.51 0.53 4 5 5 28 57 45 28 KDA PROTEIN;S-FORMYLGLUTATHIONE HYDROLASE 0.55±0.21 1.16±0.29 0.71±0.29 0.27 0.24 4 6 4 4 8 5 PROTEASOME 26S NON-ATPASE SUBUNIT 11 VARIANT (FRAGMENT) 0.90±0.42 1.89±0.65 2.38±0.61 0.19 0.21 4 6 7 12 21 26 ISOFORM 1 OF PROTEASOME SUBUNIT ALPHA TYPE 7 0.60±0.24 1.25±0.36 0.67±0.26 0.25 0.34 4 6 4 4 8 5 HEAT-SHOCK PROTEIN BETA-3 1.92±0.79 3.95±1.2 3.32±0.89 0.34 0.41 4 6 6 8 15 13 ALPHA-SOLUBLE NSF ATTACHMENT PROTEIN 0.86±0.56 1.74±0.56 1.47±0.51 0.52 0.40 3 6 5 8 14 11 UNCHARACTERIZED PROTEIN C7ORF24 1.56±0.67 3.03±1.02 1.45±0.65 0.32 0.47 4 5 4 9 14 17 ISOFORM 1 OF PHOSPHOGLUCOMUTASE-1 2.54±0.73 4.71±1.31 2.33±0.75 0.18 0.27 7 8 6 39 72 37 26S PROTEASE REGULATORY SUBUNIT 7 1.09±0.33 1.97±0.70 2.41±0.61 0.27 0.25 5 5 7 14 22 29 ISOFORM 2 OF NADH-CYTOCHROME B5 REDUCTASE 1.05±0.4 1.86±1.09 0.80±0.26 0.53* 0.92 5 4 5 10 15 6 CARDIOMYOPATHY-ASSOCIATED PROTEIN 1 0.54±0.29 0.95±0.37 1.51±0.53 0.27 0.33 4 5 6 24 44 75 81 KDA PROTEIN;SARCOPLASMIC RETICULUM HISTIDINE-RICH CALCIUM-BINDING PROTEINPRECURSOR 0.42±0.15 0.72±0.25 0.89±0.29 0.38 0.46 5 5 5 8 13 17 3-KETOACYL-COA THIOLASE, MITOCHONDRIAL 1.70±0.47 2.93±0.78 3.69±0.82 0.16 0.20 6 7 8 18 30 38 ISOFORM 1 OF PUTATIVE GTP-BINDING PROTEIN 9 0.91±0.41 1.54±0.53 1.92±0.58 0.39 0.41 4 5 5 9 16 20



110 KDA PROTEIN;KINESIN HEAVY CHAIN 0.19±0.10 0.31±0.12 0.60±0.25 0.24* 0.48 3 5 4 6 8 15 FH1/FH2 DOMAIN-CONTAINING PROTEIN 1 0.94±0.17 1.45±0.29 1.90±0.41 0.11* 0.15 7 8 7 32 44 58 UBIQUITIN CARBOXYL-TERMINAL HYDROLASE 14;UBIQUITIN SPECIFIC PROTEASE 14 ISOFORM B 0.93±0.20 1.44±0.34 1.87±0.42 0.16 0.29 7 7 8 14 18 23 CITRATE SYNTHASE, MITOCHONDRIAL PRECURSOR 1.26±0.39 1.92±0.45 2.64±1.11 0.42* 0.57 7 7 7 16 23 29 MICROSOMAL GLUTATHIONE S-TRANSFERASE 3 3.43±1.54 5.03±1.90 2.10±0.81 0.4* 0.58 4 5 4 16 20 9 AMYLO-1,6-GLUCOSIDASE, 4-ALPHA-GLUCANOTRANSFERASE ISOFORM 1 VARIANT 0.24±0.09 0.31±0.10 0.50±0.21 0.42* 0.67 5 6 6 12 12 21 21 KDA PROTEIN;TROPONIN I, FAST SKELETAL MUSCLE 8.66±3.11 10.7±5.6 17.5±9.3 0.61* 0.54 7 7 8 51 48 92 GLUTATHIONE S-TRANSFERASE MU 3 0.96±0.49 1.13±0.44 2.03±0.93 0.48 0.75 3 4 5 6 7 12 CREATINE KINASE B-TYPE 1.39±0.41 1.45±0.76 3.02±0.91 0.22 0.26 6 4 7 15 14 29 COP9 SIGNALOSOME COMPLEX SUBUNIT 8;COP9 SIGNALOSOME SUBUNIT 8 ISOFORM 2 0.69±0.27 0.68±0.33 1.49±0.53 0.26 0.48 4 3 5 4 3 7 ISOFORM 1 OF 26S PROTEASOME NON-ATPASE REGULATORY SUBUNIT 1 0.36±0.15 0.29±0.13 0.62±0.21 0.36 0.44 4 4 6 10 7 15 ENDOPLASMIN PRECURSOR 1.12±0.40 0.89±0.28 2.04±0.64 0.2* 0.32 5 6 6 24 18 43 ANNEXIN A2 ISOFORM 1 1.98±0.93 1.27±0.54 2.68±1.19 0.57 0.56 5 5 7 21 13 25 10-FORMYLTETRAHYDROFOLATE DEHYDROGENASE;FORMYLTETRAHYDROFOLATE DEHYDROGENASE ISOFORM A VARIANT 0.53±0.18 0.33±0.17 1.02±0.40 0.2 0.28 5 3 6 14 8 25



CYTOCHROME C OXIDASE SUBUNIT 5A, MITOCHONDRIAL PRECURSOR 12.8±2.46 10.3±2.85 4.49±1.70 0.06 0.10 7 6 5 60 47 20 MITOCHONDRIAL IMPORT INNER MEMBRANE TRANSLOCASE SUBUNIT TIM13 8.38±1.69 6.46±1.88 2.68±1.43 0.07 0.10 7 6 3 23 18 8 SORTING AND ASSEMBLY MACHINERY COMPONENT 50 HOMOLOG 1.13±0.42 0.83±0.30 0.40±0.26 0.32 0.29 5 5 2 14 10 5 NADH DEHYDROGENASE [UBIQUINONE] 1 ALPHA SUBCOMPLEX SUBUNIT 10,MITOCHONDRIAL PRECURSOR 0.67±0.17 0.47±0.18 0.27±0.13 0.23 0.21 6 4 3 7 4 3 60S RIBOSOMAL PROTEIN L12 1.89±0.47 1.32±0.43 0.88±0.46 0.30 0.30 6 5 3 10 6 4 ISOFORM PNS-TAU OF MICROTUBULE-ASSOCIATED PROTEIN TAU;MICROTUBULE-ASSOCIATED PROTEIN TAU 0.36±0.23 0.24±0.14 0.52±0.19 0.35 0.40 3 3 6 8 5 11 ELONGATION FACTOR 1-ALPHA 2 0.85±0.32 0.56±0.29 1.42±0.54 0.32 0.73 5 3 5 12 7 19 GUANIDINOACETATE N-METHYLTRANSFERASE 1.31±0.51 0.83±0.25 1.74±0.59 0.42* 0.50 4 5 6 10 6 11 THIOESTERASE SUPERFAMILY MEMBER 2 6.22±1.51 3.93±0.69 2.44±0.94 0.07 0.14 6 7 4 24 15 9 UBIQUITIN-CONJUGATING ENZYME E2 L3 1.99±0.58 1.25±0.54 0.44±0.29 0.1 0.12 6 4 2 8 5 2 NADH DEHYDROGENASE [UBIQUINONE] 1 BETA SUBCOMPLEX SUBUNIT 4 3.33±1.15 2.07±0.88 0.73±0.53 0.14 0.14 6 4 2 14 8 3 ISOFORM 1 OF [PROTEIN ADP-RIBOSYLARGININE] HYDROLASE-LIKE PROTEIN 1 1.47±0.55 0.72±0.3 1.56±0.30 0.29 0.20 6 4 7 16 8 15 ATP SYNTHASE B CHAIN, MITOCHONDRIAL PRECURSOR 2.21±0.61 0.99±0.48 0.89±0.50 0.18 0.19 6 4 3 18 7 6



GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE 8.82±3.27 3.51±2.22 8.85±4.86 0.5 0.33 7 4 6 102 43 95 ISOFORM 1 OF SARCALUMENIN PRECURSOR 0.32±0.14 0.10±0.07 0.27±0.09 0.29 0.21 5 2 5 9 3 7 NADH DEHYDROGENASE [UBIQUINONE] 1 BETA SUBCOMPLEX SUBUNIT 3 6.97±2.87 1.89±1.03 2.42±1.01 0.13* 0.34 5 3 4 21 5 6 ISOFORM 2 OF TROPONIN T, SLOW SKELETAL MUSCLE;ISOFORM 3 OF TROPONIN T, SLOW SKELETAL MUSCLE 1.85±0.98 0.46±0.23 1.45±0.53 0.33* 0.44 4 3 5 15 3 10 CARBONYL REDUCTASE [NADPH] 1 0.93±0.32 1.15±0.44 0.52±0.20 0.37 0.57 5 4 4 8 8 4 ISOFORM MITOCHONDRIAL OF PEROXIREDOXIN-5, MITOCHONDRIAL PRECURSOR 2.54±0.85 2.67±0.89 1.13±0.39 0.29* 0.29 6 6 5 16 16 7

Data for NSAF values are given as Means ± SEM. P value listed is for overall analysis of variance among groups. Total spectra = sum of spectra across 8 subjects in each group. *Variances not homogeneous.



4 Supplemental Table 4. Protein set analysis.

GO namespace GO name Matching Gene Names

Number of

Proteins Lean Obese Diabetic Overall

ANOVA

Molecular function

iron ion binding ACO1;NDUFS8;TF;HPX;COX5A;NDUFS2;UQCRFS1;NDUFV1;CYC1;MB;PPP1CB;SDHB;CYCS;CAT;FTH1;NDUFS1

16 1.03E±0.070 1.12E±0.071 0.840±0.070 0.03

Cellular component

mitochondrial part AFG3L2;ACAA2;TIMM13;ATP5J;NDUFA9;ACADM;COX4I1;FIS1;ATP5O;ETFA;NDUFS8;IDH2;CASQ1;NDUFA4;CPT2;UQCRC1;CHCHD3;PHB;COX2;COX5B;SOD2;NDUFAB1;HADHB;SLC25A4;UQCRQ;ATP5D;COX5A;NDUFS2;CS;NDUFS3;UQCRFS1;PHB2;NDUFV1;ATP5F1;CYC1;NDUFA10;HADHA;VDAC3;OGDH;VDAC1;ALDH4A1;NDUFA8;ATP5J2;NDUFA2;NDUFB3;NDUFA13;SLC25A11;NDUFB7;LOC727762;NDUFB4;MDH2;SDHB;ATP5B;SDHA;UQCRC2;MAOB;NNT;SLC25A12;ATP5C1;SAMM50;NDUFA6;ATP5A1;NDUFA7;CYCS;NDUFB10;UCRC;NDUFS1;SUCLG1

67 1.14±0.083 1.02±0.087 0.840±0.067 0.04

Cellular component

mitochondrial inner membrane

AFG3L2;ACAA2;TIMM13;ATP5J;NDUFA9;COX4I1;ATP5O;NDUFS8;IDH2;NDUFA4;CPT2;UQCRC1;CHCHD3;PHB;COX2;SOD2;NDUFAB1;SLC25A4;UQCRQ;ATP5D;COX5A;NDUFS2;NDUFS3;UQCRFS1;PHB2;NDUFV1;CYC1;NDUFA10;HADHA;NDUFA8;ATP5J2;NDUFA2;NDUFB3;NDUFA13;SLC25A11;NDUFB7;LOC727762;NDUFB4;SDHB;ATP5B;SDHA;UQCRC2;MAOB;NNT;SLC25A12;ATP5C1;SAMM50;NDUFA6;ATP5A1;NDUFA7;CYCS;NDUFB10;UCRC;NDUFS1;SUCLG1

54 1.16±0.081 1.02±0.098 0.821±0.072 0.03

Cellular component

mitochondrial envelope

AFG3L2;ACAA2;TIMM13;ATP5J;NDUFA9;COX4I1;FIS1;ATP5O;NDUFS8;IDH2;NDUFA4;CPT2;UQCRC1;CHCHD3;PHB;COX2;COX5B;SOD2;NDUFAB1;HADHB;SLC25A4;UQCRQ;ATP5D;COX5A;NDUFS

59 1.16±0.085 1.01±0.087 0.820±0.070 0.02



2;NDUFS3;UQCRFS1;PHB2;NDUFV1;CYC1;NDUFA10;HADHA;VDAC3;VDAC1;NDUFA8;ATP5J2;NDUFA2;NDUFB3;NDUFA13;SLC25A11;NDUFB7;LOC727762;NDUFB4;SDHB;ATP5B;SDHA;UQCRC2;MAOB;NNT;SLC25A12;ATP5C1;SAMM50;NDUFA6;ATP5A1;NDUFA7;CYCS;NDUFB10;UCRC;NDUFS1;SUCLG1

Cellular component

mitochondrial membrane

AFG3L2;ACAA2;TIMM13;ATP5J;NDUFA9;COX4I1;FIS1;ATP5O;NDUFS8;IDH2;NDUFA4;CPT2;UQCRC1;CHCHD3;PHB;COX2;SOD2;NDUFAB1;SLC25A4;UQCRQ;ATP5D;COX5A;NDUFS2;NDUFS3;UQCRFS1;PHB2;NDUFV1;CYC1;NDUFA10;HADHA;VDAC3;VDAC1;NDUFA8;ATP5J2;NDUFA2;NDUFB3;NDUFA13;SLC25A11;NDUFB7;LOC727762;NDUFB4;SDHB;ATP5B;SDHA;UQCRC2;MAOB;NNT;SLC25A12;ATP5C1;SAMM50;NDUFA6;ATP5A1;NDUFA7;CYCS;NDUFB10;UCRC;NDUFS1;SUCLG1

57 1.17±0.083 1.02±0.088 0.82±0.067 0.02

Biological process

positive regulation of apoptosis

AIFM1;TUBB2C;BCL2L13;PDIA3;NDUFS3;CUL5;NDUFA13;GPX1;PPP2R1A

9 0.820±0.081 1.27±0.13 0.90±0.10 0.01

Molecular function

transmembrane transporter activity

ATP5J;COX4I1;ATP5O;UQCRC1;COX6C;COX2;COX5B;SLC25A4;UQCRQ;ATP5D;COX5A;UQCRFS1;ATP5L;ATP5F1;VDAC3;ATP2A2;CUL5;TNNI2;VDAC1;ATP5I;ATP5J2;SLC25A11;ATP5H;ATP5B;UQCRC2;NNT;SLC25A12;ATP5C1;ATP5A1;CACNA2D1;A2M;UCRC

32 1.14±0.093 1.01±0.068 0.85±0.042 0.03

Molecular function

inorganic cation transmembrane transporter activity

ATP5J;COX4I1;ATP5O;UQCRC1;COX6C;COX2;COX5B;UQCRQ;ATP5D;COX5A;UQCRFS1;ATP5L;ATP5F1;ATP2A2;TNNI2;ATP5I;ATP5J2;ATP5H;ATP5B;UQCRC2;NNT;ATP5C1;ATP5A1;UCRC

24 1.20±0.10 0.96±0.11 0.84±0.065 0.05

Molecular function

hydrogen ion transmembrane transporter activity

ATP5J;COX4I1;ATP5O;UQCRC1;COX6C;COX2;COX5B;UQCRQ;ATP5D;COX5A;UQCRFS1;ATP5L;ATP5F1;TNNI2;ATP5I;ATP5J2;ATP5H;ATP5B;UQCRC2;N.04NT;ATP5C1;ATP5A1;UCRC

23 1.20±0.10 0.97±0.12 0.83±0.07 0.05



Molecular function

ion transmembrane transporter activity

AT.03P5J;COX4I1;ATP5O;UQCRC1;COX6C;COX2;COX5B;UQCRQ;ATP5D;COX5A;UQCRFS1;ATP5L;ATP5F1;VDAC3;ATP2A2;CUL5;TNNI2;VDAC1;ATP5I;ATP5J2;SLC25A11;ATP5H;ATP5B;UQCRC2;NNT;ATP5C1;ATP5A1;CACNA2D1;UCRC

29 1.15±0.10 0.99±0.080 0.86±0.04 0.04

Molecular function

cytochrome-c oxidase activity

COX4I1;COX6C;COX2;COX5B;COX5A 5 1.29±0.17 0.97±0.13 0.74±0.10 0.03

Cellular component

mitochondrial respiratory chain

NDUFA9;NDUFS8;NDUFA4;UQCRC1;COX2;NDUFAB1;UQCRQ;NDUFS2;NDUFS3;UQCRFS1;NDUFV1;CYC1;NDUFA10;NDUFA8;NDUFA2;NDUFB3;NDUFA13;NDUFB7;LOC727762;NDUFB4;SDHA;UQCRC2;NNT;NDUFA6;NDUFA7;CYCS;NDUFB10;UCRC;NDUFS1

28 1.21±0.11 0.99±0.12 0.80±0.09 0.04

Biological process

amino acid derivative metabolic process

P4HB;CKMT2;ALDH5A1;CKB;CKM;GAMT;PCYOX1;ALDH9A1

8 0.87±0.04 0.90±0.12 1.23±0.12 0.03

Biological process

protein amino acid phosphorylation

PRDX1;OXSR1;RPS6KA3;PRKAR1A;PRKAR2A;PHKG1;MYLK2;CAMK2B;PRKAA2;PPP2R1A;CAMK2A

11 0.79±0.08 1.06±0.10 1.14±0.10 0.04

Biological process

oxygen and reactive oxygen species metabolic process

SOD2;NDUFS3;NDUFA13;CAT;NDUFS1 5 1.09±0.05 1.12±0.09 0.79±0.07 0.01

Cellular component

mitochondrial membrane part

TIMM13;NDUFA9;FIS1;ATP5O;NDUFS8;NDUFA4;UQCRC1;COX2;NDUFAB1;UQCRQ;ATP5D;NDUFS2;NDUFS3;UQCRFS1;NDUFV1;CYC1;NDUFA10;NDUFA8;ATP5J2;NDUFA2;NDUFB3;NDUFA13;NDUFB7;LOC727762;NDUFB4;ATP5B;SDHA;UQCRC2;NNT;ATP5C1;NDUFA6;ATP5A1;NDUFA7;CYCS;NDUFB10;UCRC;NDUFS1

36 1.20±0.09 1.00±0.12 0.80±0.07 0.02

Cellular component

microtubule cytoskeleton

TUBB2C;TPT1;TUBB;KIF5B;TCP1;PPP2R1A

6 0.73±0.09 1.18±0.14 1.09±0.10 0.02

Molecular function

ubiquinol-cytochrome-c reductase activity

UQCRC1;UQCRQ;UQCRFS1;UQCRC2;UCRC

5 1.28±0.15 0.90±0.11 0.82±0.08 0.03

Cellular intrinsic to plasma VAPB;GBAS;PHB;SLC25A4;VDAC3;SLC 7 1.22±0.14 1.00±0.10 0.76±.07 0.03



component membrane 25A11;ITGB1 Biological process

regulation of transferase activity

YWHAE;CDC37;FABP4;YWHAB;YWHAG;PPP2R1A

6 0.72±0.11 1.24±0.16 1.04±0.12 0.04

Biological process

nervous system development

YWHAE;NAPA;KIF5B;ALDH5A1;RPS6KA3;BZW2;S100A6;GPI;DPYSL3;YWHAB;PFN1;RTN4;YWHAG;GSTM3;FLNC

15 0.78±0.09 1.05±0.08 1.17±0.13 0.04

Complex I NDUFA9;NDUFS8;NDUFA4;NDUFAB1;NDUFS2;NDUFS3;NDUFV1;NDUFA10;NDUFA8;NDUFA2;NDUFB3;NDUFA13;NDUFB7;LOC727762;NDUFB4;NDUFA6;NDUFA7;NDUFB10;NDUFS1

18 1.23±0.12 1.01±0.16 0.76±0.11 0.06

Complex II ALDH5A1;SDHB;SDHA 3 0.90±0.18 1.12±0.11 0.98±0.20 0.07 Complex III UQCRC1;UQCRQ;UQCRFS1;CYC1;U

QCRC2;UCRC 6 1.26±0.14 0.92±0.08 0.83±0.07 0.02

Complex IV COX4I1;COX6C;COX2;COX5B;COX5A;

5 1.29±0.17 0.97±0.13 0.74±0.10 0.03

Complex V ATP5J;ATP5O;ATP5D;ATP5L;ATP5F1;ATP5I;ATP5J2;ATP5H;ATP5B;ATP5C1;ATP5A1

11 1.16±0.12 1.03±0.19 0.81±0.10 0.22

T-Complex Protein

CCT5;CCT7;TCP1;CCT2;CCT8;CCT4 6 0.59±0.13 1.31±0.38 1.10±0.19 0.04

Data are given as Mean ± SEM (n = 8 per group) for a composite NSAF variable consisting of the average scaled NSAF values totaled across proteins in a set.



5 Supplemental Table 5. Patterns of changes among groups. Pattern Protein Direction of Change Relative to Global Median

Lean Obese Diabetic 1 STRESS-70 PROTEIN, MITOCHONDRIAL PRECURSOR ↓ ↔ ↑ 1 3-KETOACYL-COA THIOLASE, MITOCHONDRIAL ↓ ↔ ↑

1 ISOFORM LONG OF 14-3-3 PROTEIN BETA/ALPHA;ISOFORM SHORT OF 14-3-3 PROTEIN BETA/ALPHA ↓ ↔ ↑

1 ISOFORM 1 OF ADENYLOSUCCINATE LYASE ↓ ↔ ↑ 1 PROTEASOME 26S NON-ATPASE SUBUNIT 11 VARIANT (FRAGMENT) ↓ ↔ ↑ 1 PROTEIN-ARGININE DEIMINASE TYPE-2 ↓ ↔ ↑ 1 34 KDA PROTEIN.;CALPAIN SMALL SUBUNIT 1 ↓ ↔ ↑ 1 ACTIN, CYTOPLASMIC 1;ACTIN, CYTOPLASMIC 2 ↓ ↔ ↑ 1 SIMILAR TO SMOOTHELIN-LIKE 1 ↓ ↔ ↑ 1 ISOFORM 1 OF [PROTEIN ADP-RIBOSYLARGININE] HYDROLASE-LIKE PROTEIN 1 ↓ ↔ ↑ 1 ANNEXIN VII ISOFORM 2;ISOFORM 1 OF ANNEXIN A7;ISOFORM 2 OF ANNEXIN A7 ↓ ↔ ↑ 1 RETINAL DEHYDROGENASE 1 ↓ ↔ ↑ 1 PFKFB2 PROTEIN ↓ ↔ ↑ 1 RADIXIN ↓ ↔ ↑ 1 PRENYLCYSTEINE OXIDASE PRECURSOR ↓ ↔ ↑ 1 PROTEASOME SUBUNIT ALPHA TYPE 5 ↓ ↔ ↑ 1 PROTEIN S100-A4 ↓ ↔ ↑ 1 ISOFORM B OF MANNOSE-6-PHOSPHATE RECEPTOR-BINDING PROTEIN 1 ↓ ↔ ↑ 1 CULLIN HOMOLOG 5 ↓ ↔ ↑ 1 L-LACTATE DEHYDROGENASE B CHAIN ↓ ↔ ↑ 1 BIFUNCTIONAL PURINE BIOSYNTHESIS PROTEIN PURH ↓ ↔ ↑ 1 CATALASE ↓ ↔ ↑

1 CHAPERONIN CONTAINING TCP1, SUBUNIT 8 (THETA) VARIANT;T-COMPLEX PROTEIN 1 SUBUNIT THETA ↓ ↔ ↑

1 SERINE/THREONINE-PROTEIN PHOSPHATASE 2A 65 KDA REGULATORY SUBUNIT AALPHA ISOFORM ↓ ↔ ↑

1 ISOFORM 1 OF PDZ AND LIM DOMAIN PROTEIN 3 ↓ ↔ ↑ 2 GYS1 PROTEIN ↓ ↔ ↑



2 GLUTAMINYL-TRNA SYNTHETASE ↓ ↔ ↑ 2 PROTEASOME SUBUNIT BETA TYPE 3 ↓ ↔ ↑ 2 120 KDA PROTEIN.;IMPORTIN-7 ↓ ↔ ↑ 2 CARNITINE O-PALMITOYLTRANSFERASE 2, MITOCHONDRIAL PRECURSOR ↓ ↔ ↑ 2 ISOFORM 1 OF UPF0366 PROTEIN C11ORF67 ↓ ↔ ↑ 2 ISOFORM 1 OF VINCULIN.;ISOFORM 2 OF VINCULIN ↓ ↔ ↑ 2 UBIQUITIN-ACTIVATING ENZYME E1 ↓ ↔ ↑ 2 ISOFORM LONG OF UBIQUITIN CARBOXYL-TERMINAL HYDROLASE 5 ↓ ↔ ↑ 2 ENDOPLASMIN PRECURSOR ↓ ↔ ↑ 2 26S PROTEASOME NON-ATPASE REGULATORY SUBUNIT 2 ↓ ↔ ↑ 2 ISOLEUCYL-TRNA SYNTHETASE, MITOCHONDRIAL PRECURSOR ↓ ↔ ↑ 3 CALCIUM-BINDING MITOCHONDRIAL CARRIER PROTEIN ARALAR1 ↑ ↔ ↓ 3 NADH DEHYDROGENASE [UBIQUINONE] 1 ALPHA SUBCOMPLEX SUBUNIT 8 ↑ ↔ ↓ 3 ALPHA CRYSTALLIN FAMILY PROTEIN ↑ ↔ ↓

3 NADH-UBIQUINONE OXIDOREDUCTASE 75 KDA SUBUNIT, MITOCHONDRIALPRECURSOR ↑ ↔ ↓

3 CYTOCHROME C OXIDASE SUBUNIT 5B, MITOCHONDRIAL PRECURSOR ↑ ↔ ↓ 3 CYTOCHROME C OXIDASE POLYPEPTIDE VIC PRECURSOR ↑ ↔ ↓ 3 CYTOCHROME C ↑ ↔ ↓ 3 HISTONE H4 ↑ ↔ ↓ 3 ALPHA-ACTININ-2 ↑ ↔ ↓ 3 MYOZENIN-1 ↑ ↔ ↓ 3 NADH DEHYDROGENASE [UBIQUINONE] 1 ALPHA SUBCOMPLEX SUBUNIT 2 ↑ ↔ ↓ 3 ATP SYNTHASE, H+ TRANSPORTING, MITOCHONDRIAL F0 COMPLEX, SUBUNIT E ↑ ↔ ↓ 3 TROPONIN C, SLOW SKELETAL AND CARDIAC MUSCLES ↑ ↔ ↓ 3 NADH DEHYDROGENASE [UBIQUINONE] 1 BETA SUBCOMPLEX SUBUNIT 10 ↑ ↔ ↓

3 ISOFORM MITOCHONDRIAL OF PEROXIREDOXIN-5, MITOCHONDRIAL PRECURSOR ↑ ↔ ↓

3 SUPEROXIDE DISMUTASE [MN], MITOCHONDRIAL PRECURSOR ↑ ↔ ↓ 3 MITOCHONDRIAL 2-OXOGLUTARATE/MALATE CARRIER PROTEIN ↑ ↔ ↓ 3 60S RIBOSOMAL PROTEIN L38 ↑ ↔ ↓

3 NADH DEHYDROGENASE [UBIQUINONE] 1 ALPHA SUBCOMPLEX SUBUNIT 9,MITOCHONDRIAL PRECURSOR ↑ ↔ ↓



3 2,4-DIENOYL-COA REDUCTASE, MITOCHONDRIAL PRECURSOR ↑ ↔ ↓ 3 ATP SYNTHASE SUBUNIT G, MITOCHONDRIAL ↑ ↔ ↓ 4 HSP90 CO-CHAPERONE CDC37 ↓ ↑ ↔ 4 ISOFORM 1 OF LEUKOTRIENE A-4 HYDROLASE ↓ ↑ ↔

4 ISOFORM 1 OF APOPTOSIS-INDUCING FACTOR 1, MITOCHONDRIAL PRECURSOR;ISOFORM 3 OF APOPTOSIS-INDUCING FACTOR 1, MITOCHONDRIAL PRECURSOR

↓ ↑ ↔

4 DIHYDROLIPOYL DEHYDROGENASE, MITOCHONDRIAL PRECURSOR ↓ ↑ ↔ 4 PROTEIN CGI-38 ↓ ↑ ↔ 4 T-COMPLEX PROTEIN 1 SUBUNIT BETA ↓ ↑ ↔ 4 T-COMPLEX PROTEIN 1 SUBUNIT ALPHA ↓ ↑ ↔ 4 26S PROTEASOME NON-ATPASE REGULATORY SUBUNIT 3 ↓ ↑ ↔ 4 ISOFORM 1 OF ALPHA-1-ANTICHYMOTRYPSIN PRECURSOR ↓ ↑ ↔ 4 T-COMPLEX PROTEIN 1 SUBUNIT DELTA ↓ ↑ ↔ 4 PROTEIN KINASE C AND CASEIN KINASE SUBSTRATE IN NEURONS PROTEIN 3 ↓ ↑ ↔ 5 10 KDA HEAT SHOCK PROTEIN, MITOCHONDRIAL ↑ ↔ ↓ 5 NADH DEHYDROGENASE [UBIQUINONE] 1 BETA SUBCOMPLEX SUBUNIT 3 ↑ ↔ ↓ 5 THIOESTERASE SUPERFAMILY MEMBER 2 ↑ ↔ ↓ 5 ISOFORM 1 OF TROPOMYOSIN BETA CHAIN ↑ ↔ ↓

5 ISOFORM MITOCHONDRIAL OF FUMARATE HYDRATASE, MITOCHONDRIAL PRECURSOR ↑ ↔ ↓

5 23 KDA PROTEIN;FLAVIN REDUCTASE ↑ ↔ ↓

5 UBIQUINOL-CYTOCHROME C REDUCTASE COMPLEX UBIQUINONE-BINDING PROTEINQP-C ↑ ↔ ↓

5 ATP SYNTHASE O SUBUNIT, MITOCHONDRIAL PRECURSOR ↑ ↔ ↓ 5 21 KDA PROTEIN.;TROPONIN I, FAST SKELETAL MUSCLE ↑ ↔ ↓ 5 GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ↑ ↔ ↓ 5 NAD(P) TRANSHYDROGENASE, MITOCHONDRIAL PRECURSOR ↑ ↔ ↓ 6 187 KDA PROTEIN;COMPLEMENT C3 PRECURSOR (FRAGMENT) ↑ ↑ ↓ 6 APOLIPOPROTEIN A-I PRECURSOR ↑ ↑ ↓ 6 CARBONIC ANHYDRASE 1 ↑ ↑ ↓ 6 SEROTRANSFERRIN PRECURSOR.;TRANSFERRIN VARIANT (FRAGMENT) ↑ ↑ ↓ 6 APOLIPOPROTEIN A-II PRECURSOR ↑ ↑ ↓



6 CARBONIC ANHYDRASE 2 ↑ ↑ ↓

7 CDNA FLJ44241 FIS, CLONE THYMU3008436, HIGHLY SIMILAR TO 6-PHOSPHOFRUCTOKINASE, MUSCLE TYPE ↑ ↓ ↓

7 ANNEXIN A6 ↑ ↓ ↓ 7 CALSEQUESTRIN 1 ↑ ↓ ↓ 7 PHOSPHOGLYCERATE MUTASE 2 ↑ ↓ ↓ 7 TROPONIN C, SKELETAL MUSCLE ↑ ↓ ↓ 7 GUANIDINOACETATE N-METHYLTRANSFERASE ↑ ↓ ↓ 7 RAS-RELATED PROTEIN RAB-1B ↑ ↓ ↓ 7 RAS-RELATED PROTEIN RAB-2A. ↑ ↓ ↓ 7 HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN 1 ↑ ↓ ↓ 7 MALATE DEHYDROGENASE, CYTOPLASMIC ↑ ↓ ↓ 7 CYTOCHROME C1 HEME PROTEIN, MITOCHONDRIAL PRECURSOR ↑ ↓ ↓ 7 MYOSIN LIGHT POLYPEPTIDE 3 ↑ ↓ ↓ 7 GLUTATHIONE S-TRANSFERASE MU 3 ↑ ↓ ↓

7 MYOSIN REGULATORY LIGHT CHAIN 2, VENTRICULAR/CARDIAC MUSCLE ISOFORM ↑ ↓ ↓

7 CYTOCHROME C OXIDASE SUBUNIT 4 ISOFORM 1, MITOCHONDRIAL PRECURSOR ↑ ↓ ↓ 7 ISOFORM 2 OF NADH-CYTOCHROME B5 REDUCTASE ↑ ↓ ↓ 7 ALPHA-SOLUBLE NSF ATTACHMENT PROTEIN ↑ ↓ ↓ 7 HEAT SHOCK 70KDA PROTEIN 1B ↑ ↓ ↓ 7 40S RIBOSOMAL PROTEIN S3 ↑ ↓ ↓ 7 PROTEIN NIPSNAP2 ↑ ↓ ↓ 7 GLYCEROL-3-PHOSPHATE DEHYDROGENASE 1-LIKE ↑ ↓ ↓

7 SUCCINATE DEHYDROGENASE [UBIQUINONE] IRON-SULFUR SUBUNIT,MITOCHONDRIAL PRECURSOR ↑ ↓ ↓

7 ISOFORM 1 OF VESICLE-ASSOCIATED MEMBRANE PROTEIN-ASSOCIATED PROTEINB/C ↑ ↓ ↓

7 CATHEPSIN D PRECURSOR ↑ ↓ ↓ 7 PROTEASOME BETA 5 SUBUNIT ↑ ↓ ↓ 7 SUCCINATE-COA LIGASE, GDP-FORMING, ALPHA SUBUNIT ↑ ↓ ↓ 7 CARNITINE O-PALMITOYLTRANSFERASE I, MUSCLE ISOFORM ↑ ↓ ↓ 7 ENOYL-COA HYDRATASE, MITOCHONDRIAL PRECURSOR ↑ ↓ ↓



7 ISOFORM 2 OF TROPONIN T, SLOW SKELETAL MUSCLE;ISOFORM 3 OF TROPONIN T, SLOW SKELETAL MUSCLE ↑ ↓ ↓

7 MALATE DEHYDROGENASE, MITOCHONDRIAL PRECURSOR ↑ ↓ ↓

7 35 KDA PROTEIN.;GLYCEROL-3-PHOSPHATE DEHYDROGENASE [NAD+], CYTOPLASMIC ↑ ↓ ↓

7 PHOSPHOGLYCERATE KINASE 1 ↑ ↓ ↓ 7 CYTOCHROME C OXIDASE SUBUNIT 5A, MITOCHONDRIAL PRECURSOR ↑ ↓ ↓

Patterns 1-7 correspond to outlined, numbered areas on heat maps from GEDI analysis shown in Figure 3. Proteins were grouped by this analysis by the pattern of changes among the groups. ↑ = increase, ↓ = decrease, ↔ =no change, relative to a global median value.

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Online Appendix 3. Supplemental Information Methods...of at least one pair-wise comparison (obese vs. lean, diabetic vs. lean, and obese vs. diabetic). Finally, the proteins that were