The University of California San Diego (UCSD) and University of Nebraska Medical Center Institutional Review Boards (IRB) approved the studies in which samples were collected, and the UCSD and UNMC IRBs approved their use in this study. Written informed consent was received from all participants. To ascertain whether subjects might have decisional impairment, a Decisional Capacity Assessment was administered. This consisted of an 11-item post-consent quiz with questions regarding the nature of the illness being studied, the voluntary nature of participation, and the ability to withdraw at any time, the consequences of withdrawing, the possible risks and benefits of participation, the procedures involved, the time required, confidentiality, and whom to call with any questions. Assistance with reading or understanding the vocabulary was provided. Inability to achieve a perfect score on the test or other serious indication of questionable capacity resulted in further evaluation by a clinical psychiatrist, psychologist, or neurologist. All subjects in this analysis achieved a perfect score on the test, indicating satisfactory demonstration of capacity.
All participants were ambulatory and underwent evaluation in the outpatient research center at UCSD. Eligibility criteria included the ability to undergo a structured clinical interview and to provide details of combined antiretroviral therapy (cART) use and substance use history.
Data were collected according to a standardized protocol of comprehensive neuromedical, neurobehavioral, and laboratory assessments as described previously 
. Briefly, the following clinical parameters were evaluated using structured interviews and laboratory assessments where appropriate: cART use, including current and past exposure and HIV disease markers (plasma viral load and current and nadir CD4+ cell counts). Blood was collected by venipuncture and used to quantify plasma HIV viral loads by a commercial reverse transcription polymerase chain reaction assay (nominal lower quantitation limit, 50 copies/mL [Amplicor; Roche Diagnostic Systems, Indianapolis, Indiana]). Current CD4+ cell counts were measured by flow cytometry. Psychiatric diagnoses, including substance use disorders (abuse and dependence), were assessed using the computer assisted Composite International Diagnostic Interview (CIDI) 
, a fully structured clinical interview that is widely used in psychiatric research 
. The CIDI classified current (within in the last 30 days) and lifetime (>30 days ago) histories of METH use (e.g., abuse and dependence) disorders.
Ammonium phosphate, α-cyano-4-hydroxycinammic acid (CHCA), trifluoroacetic acid (TFA) were from Sigma Aldrich (St. Louis, MO, USA). HPLC grade water and acetonitrile (ACN) were from Fisher Scientific (Pittsburg, PA, USA). NuPAGE 4–12% gels were from Invitrogen Corp. (Carlsbad, CA, USA). Ready Gel™ Blotting Sandwiches and glycine were from Bio-Rad (Hercules, CA, USA). Super Signal® West Pico chemiluminescent substrate was from Pierce (Rockford, IL, USA). Mouse monoclonal antibodies against vitamin D-binding protein (Abcam, Cambridge, MA, USA), and ceruloplasmin (BD Biosciences, San Jose, CA, USA) were used as primaries, for detection of secondary antibodies were polyclonal goat HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA).
Samples had been stored at −80°C from the time of collection until the time of shipment. Samples were shipped on dry ice from UCSD to UNMC and, on arrival, remained frozen. HIV was inactivated in all samples by addition of 10 µl of 10% Triton-X100 freshly made and 50 µl of cocktail of protease inhibitors (Sigma-Aldrich St. Louis, MO) per 1 mL of sample. After 30 minutes samples were aliquoted and those unused were stored at −80°C. Two hundred fifty microliters from each sample were filtered using 0.2 µm spin filter and immunodepleted using an IgY14 column (Sigma-Aldrich) connected to a liquid chromatography systems HPLC to immunodeplete abundant plasma proteins: albumin, α1-antitrypsin, IgM, haptoglobin, fibrinogen, α1-acid glycoprotein, apolipoprotein A-I and A-III, Apolipoprotein B, IgG, IgA, transferrin, α2-macroglobulin and complement C3. Flow-through fractions containing unbound proteins were concentrated using Vivaspin 15R (Sartorius, Aubagne, France), according to the manufacturer protocol. Finally, protein concentration was determined with a NanoDrop spectrophotometer (Thermo Scientific, San Jose, CA).
Trypsin digest and peptide labeling
Fifty micrograms of proteins were precipitated with ethanol. Briefly, we added 10 volumes of cold ethanol (200 proof) to each sample. Samples were incubated for 3 h at −20°C and centrifuged at 13000× g for 15 minutes at 4°C. Proteins pellets were washed with 1 ml of 70% ethanol and dried in SpeedVac (ThermoScientific). Subsequent solutions were provided with iTRAQ reagent kit (Applied Biosystem (ABI), Carlsbad, CA).
Dried proteins were solubilized with dissolution solution and proteins were denatured with 1 µl of denaturant reagent. Proteins reduction with reducing reagent was performed for 1 h at 60°C and finally cysteine blocking solution was used to block cysteines during 10 minutes at room temperature.
Trypsin from ABI was reconstituted at 1 µg/µl with milliQ water and 10 µg of trypsin were added to each sample. Digestion was performed for 16 h at 37°C.After digestion, peptides were labeled with iTRAQ label reagent (ABI) at room temperature for 2 h and 8 samples, labeled with the different isobaric mass tags, were combined in one tube and dried with SpeedVac.
iTRAQ labeled peptides processing
Having subsequent samples collected from the same individuals at various times allowed us to make comparisons between time points as well as across all groups. Because we had four groups and two time points per group, the 8-plex iTRAQ approach to quantify changes was the method of choice. shows how samples were “scrambled” into four sets to remove tag labeling bias. Since Group 1, (HIV+/persistent METH+) had only four individuals, four samples were randomly chosen from those available in Groups 2, 3 and 4 (consisting of 8, 12 and 10 identified subjects, respectively). One set of samples utilized all eight iTRAQ tags and each was measured in a separate analytical run.
iTRAQ tag assignation for 4 biological replicates in experimental design.
Samples were clarified using mix education exchange (MCX) column (Water Corp., Milford, MA). Label peptides were solubilized with 1 ml of 0.1% formic acid, passed through the column, then the column was first washed with 5% methanol, 0.1% formic acid solution and then with methanol HPLC grade. Peptides were eluted with 1.4% NH4
OH in methanol 
Samples were dried, reconstituted in 1.44 ml of 0.1% formic acid and supplemented with 1.44 µl of OFFGEL solution for 360 µl of peptides. Next, samples were fractionated based on their isolelectric point (pI) using 3100 OFFGEL Fractionator (Agilent, Inc. Santa Clara, CA). OFFGEL strips were rehydrated for 15 minutes at room temperature with 40 µl of OFFGEL solution. Peptide samples were loaded onto strips, splitting them equally between all 12 wells. Separation was performed for 20,000 Vhrs.
Collected fractions were cleaned with C-18 spin columns, according to the manufacturer's protocol. Briefly, fractions were adjusted to 5% acetonitrile (ACN) and 0.5% trifluoric acid (TFA) and passed through activated columns. Columns were washed twice with a 5% ACN, 0.5% TFA solution and peptides were eluted with a 70% ACN, 0.1% TFA solution. Peptides were finally dried and stored at −80°C until further use.
Off line LC-MS/MS analysis
Subsequent fractionation of OFFGEL fractions was performed off-line using Tempo™ LC system with automatic high density spotting onto MALDI target plates. Peptides were solubilized in 20 µl of 0.1% TFA and 10 µl of samples were loaded onto a ProteoCol™ C18 trap cartridge (MichromBiosources, Auburn, CA) and washed for 20 minutes at 9 µl/min. Gradient of separation was realized using a ratio between two buffers, Water
0.1) (Buffer A) and water
0.1) (Buffer B). To perform the separation, the subsequent gradient was applied, time 0 to 5 min 5 to 15% buffer B, 5–52 minutes 15–35%, 52–54 minutes 35–80%, 54–64 minutes 80%, 64–65 minutes 80—5% and 65–72 minutes 5%. Peptide elution was monitored with a UV cell at 214 nm absorbance. After the UV cell, eluted peptides were mixed with a matrix solution (1.2 mg/ml in 75% ACN and 0.1% TFA solution) at a flow rate 1 µl/minute using a Harvard Apparatus syringe pump. Fractions were spotted every 30 seconds and voltage applied to the plate during spotting was 2.8 kV.
Spotted fractions were submitted for data acquisition on a 4800 MALDI-TOF/TOF mass spectrometer (ABI). MS spectra were acquires from 800 to 3000 m/z, for a total of 1000 laser shots by an Nd-YAG laser operating at 355 nm and 200 Hz. Laser intensity remain fixed for all the analyses. MS/MS analyses were performed using 1 kV collision energy with air as CID gas. Metastable ions were suppressed, for a total of 1000 laser shots.
Protein identification and quantification were performed with ProteinPilot™ software using Paragon method. The search parameters were as follows: iTRAQ 8plex (peptide labeled), Methylthio alkylation of Cysteine, NCBI database restricted to Homo sapiens.
Protein/peptide abundance measures generated by iTRAQ were first pre-processed using ProteinPilot™ v2.0. The logarithm of the abundance measure was modeled as a function of animal, protein, peptide, tag and experimental condition. Four sets of samples were used to simultaneously collect data from eight experimental conditions composed of two visits from four groups: Group 1: HIV+/persistent METH+, Group 2: HIV+/ST METH abstinent, Group 3: HIV+, LT METH abstinence, Group 4: HIV-/METH-. All available abundance records (N
77,159) were normalized using an iterative back fitting procedure to remove the animal, protein and peptide effects. Comparison of the distribution of the protein/peptide normalized (log) abundance measures by experimental condition was restricted to records with ProteinPilot™ assessed “confidence” of at least 50%. The mixed model was fit on the normalized abundances of each protein to adjust for the correlation of the samples collected from the same subject. The abundances of each protein between two visits within the same group were compared, and the relative abundance and the p-values were calculated to evaluate the short/long term effects of METH within each group.
Western blot - validation
Western blot quantitation was done as previously described 
. 1DE was performed using NuPAGE gel system (Invitrogen Corp.) in 4–12% gradient Bis-Tris gels under reducing conditions and transferred onto PVDF membrane. Chemiluminescent signal was detected using SuperSignal West Pico™Chemiluminescent Substrate and recorded on Blue Lite X-ray film (ISCBioExpress, Kaysville, UT).