ABSTRACT
Rare earth elements (REE) can produce M2+ ions in ICP-MS and 150Nd2+, 150Sm2+, and 156Gd2+ can produce false positives on 75As and 78Se. Alternative instrumental tuning conditions, that utilize lower He flows within the collision cell, reduce these false positives by a factor of 2 (to 0.8 ppb As and 19 ppb Se in solutions containing 50 ppb Nd and Gd) with comparable 16O35Cl+ reduction (<100 ppt false 51V in 0.4% HCl) and Se sensitivity (DL < 1 ppb). Further reduction of these false positives is achieved by estimating the M2+ correction factors and utilizing them in the interference-correction software. Approaches to estimating the M2+ correction factor were evaluated with an emphasis on techniques that tolerate daily variability in end-user backgrounds and their ability to reduce the initial and ongoing purity requirements associated with the rare earth standards used to estimate the M2+ correction factor. The direct elemental and polyatomic overlaps associated with unit-mass approaches tend to overcorrect as non-rare-earth signals as small as 30 cps at the unit mass can induce bias relative to the <300 cps signals associated with the M2+ from a 50 ppb REE standard solution. Alternatively, shifting the M2+ estimate to a half mass (i.e., m/z 71.5: 143Nd2+) eliminates the direct overlap source of bias and allows the unit mass signal to approach 150000 cps before it bleeds over on the 1/2 mass because of abundance sensitivity limitations. The performance of the half-mass approach was evaluated in reagent water and regional tap waters fortified with Nd, Sm, and Gd at 2 ppb and 50 ppb. In addition, a half-mass in-sample approach was also evaluated. This approach was found to be beneficial relative to the external or fixed-factor half-mass approach as it could compensate for instrument drift and matrix-induced shifts in the M2+ factors. Finally, all results were evaluated relative to the As and Se concentrations determined using an ICP-QQQ in mass shift mode and a high-resolution ICP-MS.
ABSTRACT
This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body's responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697-710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma-mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 µm glass beads in a 1% Triton x-100 lysis buffer (V, T) was found to be most advantageous to extract intact zinc metalloproteins as demonstrated by the highest Zn to protein ratio, 1.030 ng Zn/µg protein, and Zn distribution among high, mid, and low molecular weights suggesting the least amount of protein denaturation. Graphical abstract In this work, several cell lysis techniques and two lysis buffers were investigated to evaluate the preservation of the zinc metalloproteome of H. capsulatum while maintaining compatibility with the analytical techniques employed.
