Introduction

Protein glycosylation is the most common posttranslational modification of proteins that are destined for extracellular environments (1), and it has been shown that glycosylation plays a critical role in protein interactions, folding, protein stabilization, subcellular localization, and protein function (2). Typically, carbohydrates are linked to the side chains of serine or threonine residues (O-linked glycosylation) or to asparagine residues (A-linked glycosylation) (2). A-linked...

Sample Preparation

Solubilization With Lysis Buffer for Minimal Dyes 1. Lysis labeling buffer 7 Murea, 2 Mthiourea, 4 (w v) CHAPS (Calbiochem), 30 mM Tris (pH 8.5) at 4 C. Check that the final solution pH is 8.5 at 4 C, to ensure good labeling efficiency. Dispense into approx 1-mL aliquots and store at -80 C, stable for several weeks. 2. Quenching solution for minimal CyDyes 10 mMl-lysine in water. 3. Rehydration buffer 7 M urea, 2 M thiourea, 4 (w v) CHAPS, 2 mg mL dithio-threitol (DTT), 1 Pharmalyte...

Data Preprocessing

Global alignment to circumvent the slight variation in m z values for the same relative data points in different spectra, reflectron data points are numbered consecutively by assigning the observed m z value that is closest to the expected MH+ for the C12 isotope of bradykinin, which is 1060.569, as data point zero. Linear data points are numbered consecutively by assigning the observed m z value that is closest to the expected average MH+ of oxidized insulin chain B, which is 3496.95, as data...

Materials

Vial of both C12 (light) and C13 (heavy) ABI Cleavable ICAT reagent (cat. no. 4339038). 2. ABI cleaving reagents A and B (supplied with ABI Cleavable ICAT reagent, cat. no. 4339038). 3. 100 g of control and experimental protein sample. 4. High-resolution cation-exchange column (PolySulfoethyl A Column, 5 im 300 A bead, from PolyLC, Inc., 2.1 x 200 mm, cat. no. 202SE0503). 5. Sequencing-grade trypsin (100 g, Promega cat. no. V5280). 6. Electrospray ionization mass spectrometer with tandem MS...

Assigning Mass Spectra to Protein Sequences

Enter the universal resource locator (URL) of a GPM server, e.g., http h.thegpm.org. The main user data input page (Fig. 1) should appear in the browser window (see Notes 1 and 2). Fig. 1. The Global Proteome Machine experimental data query interface. Each section of the search form can be expanded (or hidden) by clicking on the appropriate + symbol next to the section header. Fig. 1. The Global Proteome Machine experimental data query interface. Each section of the search form can be expanded...

Ewa Folta Stogniew

Size-exclusion chromatography (SEC), coupled with on-line static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Such glycosylated proteins or protein-detergent complexes show anomalous behavior on SEC, thus presenting a challenge to determination of...

Spot Picking Tryptic Digestion

The protein spot pick list is transferred to the Ettan Spot Picker instrument (Amersham Biosciences GE), which automatically excises the selected protein spots (using a 2.0 mm diameter pick head) from the gel and transfers them into a polypropylene 96-well microtiter plate (Corning, Corning, NY) (see Note 19). 2. The excised protein spots are then subjected to automated in-gel tryptic digestion on the Ettan TA Digester, or manually digested using the following steps a. Wash the spots with 2 x...

Antibody Microarrays Using Resonance Light Scattering Particles for Detection

Geierstanger, Petri Saviranta, and Achim Brinker Antibody microarray measurements show great potential for the simultaneous quantification of many proteins in small amounts of body fluids and extracts. Over the last few years, a micro-array platform centered around the concept of microarrays in microtiter wells was developed, and for the best assays we have achieved lower limits of detection in the femtomolar range using resonance light-scattering particles for the staining of...

OnChip Protein Synthesis for Making Microarrays

Target Protein Array

Niroshan Ramachandran, Eugenie Hainsworth, Gokhan Demirkan, and Joshua LaBaer Protein microarrays are a miniaturized format for displaying in close spatial density hundreds or thousands of purified proteins that provide a powerful platform for the high-throughput assay of protein function. The traditional method of producing them requires the high-throughput production and printing of proteins, a laborious method that raises concerns about the stability of the proteins and the shelf life of the...

Microcapillary Column Construction and Sample Loading

Currently, all samples are desalted on-line using columns similar to the three-phase microcapillary columns described in McDonald et al. ( ). These columns contain reversed-phase material, followed by strong cation exchange material, followed by reversed-phase material. In an abbreviated fashion, they are RP SCX RP columns. By using these columns, one does not need to carry out additional sample cleanup and buffer exchange prior to loading. For sample quantities of 400 pg or less, the...

Multidimensional Chromatography and Tandem Mass Spectrometry

Mudpit Mass Spectrometry

The MudPIT system we use is a combination of the Agilent1100 quaternary pump stack with thermostatted autosampler, LCQ DECA-XPplus tandem mass spectrometer, and a nanoelectrospray stage that interfaces the two systems. Furthermore, the hand-made microcapillary columns described above are single use, and the fused silica portion of each column is discarded after every analysis see Note 6 . 3.3.1. Setup of the Nanoelectrospray Stage 1. A detailed schematic of the nanoelectrospray stage is shown...

Derivatization of Affinity Pipets With Antibody

All materials for the derivatization of the affinity pipets are made ready the anti-CYSC antibody solution is diluted 1 100 in the ligand-coupling buffer and dispensed into individual wells of a 96-well microplate in 100- L aliquots the ETA, hydrochloric acid, and reconditioning buffer solutions are also dispensed into 96-well microplates, but at 200 L per well. 2. The underivatized affinity pipets are loaded onto the robotic workstation along with reagent microplates Fig. 1 . 3. The loaded...

Determination of the Oligomeric State of the Sample Protein

Filter the protein sample through a 0.22-pm low protein-binding filter. 2. Inject an appropriate amount of sample see Table 1 for optimal amounts of sample and record signals from all three detectors an example of three-detector monitoring is shown in Fig. 1 . 3. Compute the extinction coefficient, A, for the protein analyzed using Eq. 6. 4. Baseline-correct the signal from all three detectors. 5. Measure volume or height of the UV, LS, and RI peaks. 6. Calculate molar mass of the...

Martijn Verdoes Celia R Berkers Bogdan I Florea Paul F van Swieten Herman S Overkleeft and Huib Ovaa

Proteolysis is a key mechanism for protein homeostasis in living cells. This process is effected by different classes of proteases. The proteasome is one of the most abundant and versatile proteases, bearing three different proteolytic active sites. The proteasome plays an important role in essential biological pathways such as antigen presentation, signal transduction, and cell-cycle control feedback loops. The aim of this work is to design novel chemical strategies for capturing, detection,...